Effects of Long-Term Sample Preservation on Flow Cytometric Analysis of Natural Populations of Pico- and Nanophytoplankton

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1 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 MITSUHIDE SATO*, SHIGENOBU TAKEDA and KEN FURUYA Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo , 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 and Prochlorococcus, and weakened in nanoeucaryotes. Cell loss was not significant except for. Caution is required when analyzing flow cytometric data of samples stored for more than a month. Keywords: Flow cytometry, phytoplankton, preservation, natural community. 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. aa57075@mail.ecc.u-tokyo. ac.jp Copyright The Oceanographic Society of Japan/TERRAPUB/Springer 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, 903

2 A B Red fluorescence C D E 6-µm beads 2-µm beads 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 12- liter 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, 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 ( 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- 904 M. Sato et al.

3 Red fluorescence A B Prochlorococcus C 6-µm beads Red fluorescence (relative intensity to unfixed) (St. 7) (St. 7) (St. 10) Prochlorococcus 2-µm beads 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, 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 was too low to form a distinct cluster. Thus, 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 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. 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, 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. 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 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 reported by Vaulot et al. Effects of Long-Term Sample Preservation on Flow Cytometric Analysis 905

4 Orange fluorescence (relative intensity to unfixed) Side light scatter (relative value to unfixed) 2 1 (St. 7) (St. 7) (St. 10) Fig. 4. Changes in intensity of cellular orange fluorescence (FL2) of 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. 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. Forward light scatter (relative value to unfixed) (St. 7) (St. 7) (St. 10) 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. Cell counts (relative value to unfixed) (St. 7) (St. 7) (St. 10) * * 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 Cavender- Bares (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- 906 M. Sato et al.

5 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 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, 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 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

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