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Long-term preservation of Tetraselmis indica (Chlorodendrophyceae, Chlorophyta) for flow cytometric analysis: Influence of fixative and storage temperature
Journal of Microbiological Methods 139 (2017) 123–129
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Long-term preservation of Tetraselmis indica (Chlorodendrophyceae, Chlorophyta) for flow cytometric analysis: Influence of fixative and storage temperature
MARK
Sangeeta Mahableshwar Naik, Arga Chandrashekar Anil⁎ Academy of Scientific and Innovative Research (AcSIR), CSIR-National Institute of Oceanography (NIO), Dona Paula 403 004, Goa, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Tetraselmis indica Flow cytometry Glutaraldehyde Storage temperature Chlorophyll autofluorescence
Immediate enumeration of phytoplankton is seldom possible. Therefore, fixation and subsequent storage are required for delayed analysis. This study investigated the influence of glutaraldehyde (GA) concentrations (0.25%, 0.5%, and 1%) and storage temperatures (−80 °CLN2, −80 °C, −20 °C, and 5 °C) on Tetraselmis indica for flow cytometric analysis. Cell recovery, granularity, and membrane permeability were independent of GA concentration whereas cell size and chlorophyll autofluorescence were concentration dependent. After an initial cell loss (16–19%), no cell loss was observed when samples were stored at 5 °C. Cell recovery was not influenced by storage temperature until 4 months but later samples preserved at −80 °CLN2, −80 °C, and −20 °C resulted in ~41% cell loss. Although maximum cell recovery with minimal effect on cell integrity was obtained at 5 °C, autofluorescence was retained better at − 80 °CLN2 and −80 °C. This suggests that in addition to fixative, the choice of storage temperature is equally important. Thus for long-term preservation, especially to retain autofluorescence, the use of lower concentration (0.25%) of GA when stored at a lower temperature (−80 °CLN2 and − 80 °C) while a higher concentration (1%) of GA when stored at a higher temperature (5 °C) is recommended.
1. Introduction Phytoplankton are the foundation of marine food web dynamics, in spite of their small size, on which relies most of the aquatic life forms. In addition to their role in biogeochemical cycles, they also contribute to 40% of the global primary productivity (Falkowski, 1994). Considering they are important producers in the world's estuarine and coastal waters (Cloern et al., 2014), any shift in their community structure can alter the trophodynamics. Traditionally, phytoplankton were enumerated using microscopy but with the advent of automated technology such as flow cytometry (FCM); cell counting is faster, efficient, precise, and prone to minimal errors. FCM not only enables cell counting and sorting (e.g. Marie et al., 2017), but also facilitates cell cycle analysis and simultaneous measurements of multiple cellular parameters (cell size, cell granularity, autofluorescence, and fluorochrome-induced fluorescence) of an individual cell (Marie et al., 2005). Although phytoplankton studies are recommended in the real time i.e., fresh and unfixed, it is not always a feasible approach. Thus, fixation of samples and subsequent storage is mandatory. The effect of fixation and preservation on phytoplankton has been evaluated in
several studies, both for laboratory cultures and natural population (Katano et al., 2009; Marie et al., 2014; Naik et al., 2010; Pan et al., 2005; Troussellier et al., 1995). However, fixation of fragile organisms such as prasinophytes and cryptomonads engenders significant cell loss and alters cellular characteristics (Murphy and Haugen, 1985). A universal protocol of fixation and storage for all phytoplankton is difficult. The best way to overcome this problem is to optimize the protocol based on the target phytoplankton, aim, and duration of the study. Numerous fixatives are available for phytoplankton studies, yet not all are suitable for FCM analysis. For FCM analysis, the fixative used must allow the cells to remain in single-cell suspension. Single-cell analysis has been recognized as an important technology to elucidate key cellular functions which are otherwise difficult from bulk measurements (Fritzsch et al., 2012). Likewise, the chlorophyll autofluorescence is also a pre-requisite for the distinction between autotrophic and heterotrophic phytoplankton (Bloem et al., 1986; Navaluna et al., 1989). As a result, an ideal fixative should preserve both these properties of the cells. Paraformaldehyde (PFA) and glutaraldehyde (GA) are the most popular and effective fixatives used for FCM analysis of both natural population and laboratory cultures of phytoplankton (e.g. Katano et al., 2009; Marie et al., 2014, 2005; Sato et al., 2006;
Abbreviations: CV, coefficient of variation; FCM, flow cytometry; GA, glutaraldehyde; PBS, phosphate-buffered saline; PFA, paraformaldehyde ⁎ Corresponding author at: CSIR-National Institute of Oceanography, Dona Paula 403 004, Goa, India. E-mail address: [email protected] (A.C. Anil). http://dx.doi.org/10.1016/j.mimet.2017.05.018 Received 25 April 2017; Received in revised form 29 May 2017; Accepted 29 May 2017 Available online 30 May 2017 0167-7012/ © 2017 Elsevier B.V. All rights reserved.
Journal of Microbiological Methods 139 (2017) 123–129
S.M. Naik, A.C. Anil
temperature (n = 3 replicates × 5 sampling days = 15).
Vaulot et al., 1989) because they preserve both cellular structures and pigment autofluorescence. In this study, Tetraselmis indica was used as a model organism. Tetraselmis species were traditionally regarded as the members of Prasinophyceae but recent work has proven their relationship with the core Chlorophyta (Arora et al., 2013; Leliaert et al., 2012). Tetraselmis indica was isolated from a saltpan of Goa where salinity and temperature ranged from 35 to 350 and 28 to 48 °C respectively (Arora et al., 2013). Tetraselmis species even have implications in ballast water management programs (e.g. Carney et al., 2013). Due to such extreme environment of saltpans, this alga has to possess a remarkable physiology to adapt to these conditions. Our recent work on T. indica has revealed their interesting physiology by adapting to 6 months of darkness and retaining the growth potential (Naik & Anil, unpublished). Therefore, to study the physiological adaptation of T. indica to various environmental conditions, a suitable fixation and storage protocol is desirable. Classical fixatives such as Lugol's iodine, formalin, and methanol have been frequently used to fix and preserve Tetraselmis species (e.g. Azma et al., 2011; Carney et al., 2013; Ozbay and Jackson, 2010). The problem with these fixatives is that Lugol's and formalin modify the cell shape and affect the fluorescence whereas alcohol (methanol, ethanol) extracts the lipophilic pigments thereby causing the loss of chlorophyll autofluorescence (Marie et al., 2005). Owing to this, these fixatives cannot be used for FCM analysis. Fixing T. indica with PFA prepared using phosphate-buffered saline (PBS-PFA) resulted in the formation of cell aggregates which failed to meet the first criterion for FCM analysis i.e., single-cell suspension. Therefore, GA was the next fixative of choice. In view of this, the objective of this study was to evaluate the 1) influence of GA concentration on cell recovery and cellular parameters of T. indica and 2) role of storage temperature in long-term preservation of T. indica.
2.3. FCM analysis Sample analysis was performed using FACS Verse flow cytometer (BD Biosciences- US) equipped with a blue laser (488 nm). The data acquisition was triggered on red fluorescence (700/54 nm). The day-today cytometer performance and consistency were maintained and standardized using BD FACSSuite™ CS & T research beads. Forward scatter (a proxy for cell size), side scatter (a proxy for cell granularity), red fluorescence (a proxy for chlorophyll autofluorescence), and green fluorescence (SYBR Green I induced) was recorded in list mode and processed using FACS Suite software. Fluoresbrite® YG microspheres of size 10.3 μm (Polysciences Co., USA) were added as an internal standard to compare the cellular parameters of different samples. The normalization of cellular parameters was done by dividing the median value of cell population with the median value of beads (Marie et al., 2014). The effect of fixative and storage temperature on green fluorescence (a proxy for cell membrane permeability) was analyzed by staining the samples with 10 μM (final concentration) SYBR Green I (Invitrogen) followed by dark incubation for 1 h. For each sample, 10,000 events were recorded. 2.4. Statistical analyses The normality and homogeneity of the data were determined using the Shapiro Wilk's and Levene's test to meet the assumptions of parametric tests. The variation in various parameters with days was carried using one-way analysis of variance and Tukey's post hoc test was applied. All the statistical analyses were performed using the SPSS statistical package (Windows Version 22). 3. Results
2. Material and methods 3.1. Effect of GA concentrations 2.1. Culture conditions After an initial cell loss (16–19%) on fixing (p < 0.001, Tukey's post hoc test) due to cell death or lysis, insignificant cell loss occurred later (p > 0.05, Tukey's post hoc test; Fig. 1A). This indicates that the cell recovery was independent of the GA concentration tested. However, a significant change in cell size, granularity, autofluorescence, and membrane permeability was observed immediately after fixation compared to the unfixed samples of day 0 (15 min) (p < 0.001, Tukey's post hoc test; Fig. 1B–E). The addition of GA increased the cell size but decreased the granularity, autofluorescence, and membrane permeability (p < 0.001, Tukey's post hoc test). The increase in cell size was maximum in 1% (44.88%) fixed samples followed by 0.5% (39.38%) and 0.25% GA (29.68%) (p < 0.001, Tukey's post hoc test). The change in cell granularity was influenced by the GA concentration initially; with insignificant change in 0.25% fixed samples (p > 0.05, Tukey's post hoc test) and maximum change in 1% fixed samples (p < 0.001, Tukey's post hoc test). Later, insignificant variations were observed and it was independent of GA concentrations (p > 0.05, Tukey's post hoc test). At the end of 1 month, the autofluorescence was reduced by 15.13% in 1% fixed samples followed by 0.5% (23.16%) and 0.25% (28.73%). Apart from the initial fluctuations (till day1), the membrane permeability was independent of GA concentrations (p > 0.05, Tukey's post hoc test). Interestingly, the membrane permeability was lower for fixed samples compared to the unfixed samples.
Tetraselmis indica was maintained in f/2 without silicate enriched natural seawater (salinity 35) at 25 °C ± 2 °C (Guillard and Ryther, 1962). The light intensity of 40 μmol photons m− 2 s− 1 was provided by daylight white fluorescent tubes with a photoperiod of 12:12 light:dark cycle. The culture flasks were gently shaken by hand every day to ensure homogenous mixing. 2.2. Experimental design The effect of GA concentration was assessed on the exponentially growing (8-days) culture of T. indica. A control of three individual replicates without fixation was analyzed on day 0 while the remaining samples were stored in dark at 5 °C (n = 3 replicates × 5 sampling days = 15). Samples (1.5 mL) fixed with three concentrations of GA (Sigma; electron microscopy grade; final concentration: 0.25%, 0.5%, and 1%) were incubated in dark for 15 min at room temperature. One triplicate set was analyzed immediately (15 min) after fixation while the other sets were stored in dark at 5 °C (n = 3 replicates × 5 sampling days = 15). After 1 day, 7 days, 13 days, and 1 month; samples were thawed at room temperature before FCM analysis. The percentage of cell recovery was obtained with reference to unfixed samples of day 0 (labelled as control- 15 min). To understand the role of storage temperature in cell recovery and preservation of cellular parameters, fixed (0.25% GA) as well as unfixed samples were stored at four different temperatures: 1) −80 °CLN2, 2) − 80 °C, 3) −20 °C, and 4) 5 °C. In − 80 °CLN2 treatment, eppendorf tubes containing samples were immersed in liquid nitrogen followed by storage at − 80 °C. Samples were thawed and analyzed after 1 day, 1 month, 2 months, 4 months, and 6 months of storage at the respective
3.2. Effect of storage temperatures The cell recovery was independent of GA concentrations tested. As a result, to elucidate the influence of storage temperature on cell recovery and cellular parameters, four storage temperatures were tested. For this purpose, 0.25% GA concentration was used. 124
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Fig. 1. Effect of glutaraldehyde concentrations (0% (control), 0.25%. 0.5% and 1%) on Tetraselmis indica stored at 5 ºC for different time interval. A) Percentage of cell recovery, B) Forward scatter (a proxy for cell size), C) Side scatter (a proxy for cell granularity), D) Red fluorescence (a proxy for chlorophyll autofluorescence), and E) Green fluorescence (a proxy for cell membrane permeability). Percentage of cell recovery was obtained with reference to unfixed samples of day 0 (labelled as control-15 min). The dotted line indicates the normalized value of day 0 unfixed samples prior to fixation and storage. Values are mean ± SD (n = 3).
by 34.6% within 1 day in samples stored at 5 °C. Although it decreased at all storage temperatures tested, the maximum reduction of 41.43% was observed in samples stored at 5 °C for 6 months. The samples stored at − 80 °C and − 80 °CLN2 showed the best autofluorescence retention with only 18–23% reduction after 6 months. The membrane permeability was independent of storage temperatures (p > 0.05, Tukey's post hoc test) except for 5 °C initially and at − 20 °C later (Fig. 2E).
The cell recovery was almost constant at all storage temperatures till 4 months (p > 0.05, Tukey's post hoc test; Fig. 2A). Later, ~ 41% cell loss was observed at − 80 °CLN2, − 80 °C, and − 20 °C storage temperatures (p < 0.001, Tukey's post hoc test) while at 5 °C the change was negligible (p > 0.05, Tukey's post hoc test). After 6 months, the cell size in −80 °CLN2 and 5 °C stored samples was almost same and relatively similar to the unfixed samples (p > 0.05, Tukey's post hoc test) compared to other storage temperatures (Fig. 2B). The cell granularity did not show significant variation at −80 °CLN2, −80 °C, and 5 °C storage temperature (p > 0.05, Tukey's post hoc test) except for − 20 °C (p < 0.001, Tukey's post hoc test; Fig. 2C). The autofluorescence retention was significantly different at all storage temperatures (p < 0.001, Tukey's post hoc test; Fig. 2D) and was reduced
4. Discussion Enumeration of phytoplankton is a tedious process and therefore immediate analysis is seldom possible. Consequently, fixation is indispensable to avoid deterioration of samples for delayed FCM or 125
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Fig. 2. Effect of storage temperatures (− 80 °CLN2, − 80 °C, − 20 °C, and 5 °C) on Tetraselmis indica fixed with 0.25% glutaraldehyde for the different time interval. A) Percentage of cell recovery, B) Forward scatter (a proxy for cell size), C) Side scatter (a proxy for cell granularity), D) Red fluorescence (a proxy for chlorophyll autofluorescence), and E) Green fluorescence (a proxy for cell membrane permeability). Percentage of cell recovery was obtained with reference to unfixed samples of day 0 (labelled as control- 15 min). The dotted line indicates the normalized value of day 0 unfixed samples prior to fixation and storage. Values are mean ± SD (n = 3).
single-cell suspension and chlorophyll autofluorescence retention. Despite PFA being one of the popular choices for FCM analysis, in this study, PBS-PFA resulted in the formation of cell aggregates which was observed microscopically. This could be due to the extrusion of cellular material and enhanced scattering due to PFA (Navaluna et al., 1989). For this reason, PFA was regarded unsuitable for the fixation of T. indica. Precipitation on using PBS-PFA has also been reported in earlier study (Katano et al., 2009). Since GA is stable over time, commercially available, and less toxic than PFA (Marie et al., 2014); it was the next preferred choice. GA is also known to maintain the biomass of preserved plankton (Kimmerer and McKinnon, 1986), easily and rapidly cross-links proteins, and inactivates enzymes during fixation than other aldehydes (Hopwood, 1967).
microscopic analysis. Fixation is also needed to enhance the cell membrane permeability (Navaluna et al., 1989) for molecular biology studies (e.g. immunofluorescence, cell cycle analysis) as well as for microscopic examination of phytoplankton. A fixative can be an ideal choice for one organism but a bad choice to another organism. This is because different phytoplankton cultures react differently to fixation (Marie et al., 2014). An ideal fixative should preserve cells against the microbial activity, osmotic damage, autolysis, and maintain an accurate structure representation of the living cells (Jones, 1976). The objective of this study was to develop a preservation protocol for FCM analysis of T. indica and obtain maximum cell recovery with no or least effect on the cellular parameters. The obligatory attributes for FCM analysis which a fixative must possess are an ability to maintain 126
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when stored at a lower temperature (− 80 °CLN2 and −80 °C) and a higher concentration of GA (1%) when stored at a relatively higher temperature (5 °C) is recommended to retain the autofluorescence. The autofluorescence retention can also depend on the physiological status, taxonomic groups, and thawing (Bloem et al., 1986; Sato et al., 2006; Taylor and Fletcher, 1998). In this study, healthy and exponentially growing cells were used. Therefore, the impact of the chemical fixation or storage temperature due to the physiological status of cells can be ignored. Studies carried out on picoplankton population fixed with 2% GA (Hall, 1991) and heterotrophic- and phototrophicnanoflagellates fixed with 1% GA (Bloem et al., 1986) could not retain the autofluorescence satisfactorily. On the other hand, cultured Chlorella species and Haematococcus pluvialis, as well as green algae and cyanobacteria from field samples demonstrated longer retention of autofluorescence (Bloem et al., 1986). The authors attributed this difference in the autofluorescence retention to be dependent on the type of organism under study. However, they did not comment on the role of storage temperature. Additionally, phytoplankton are subjected to series of chemical and physical stress during freezing, low-temperature storage, and thawing; which may result in loss of cell viability (Taylor and Fletcher, 1998) and eventually autofluorescence. Regardless of the fact that the cell autofluorescence is used as a proxy for chlorophyll autofluorescence, care must be taken before relying on it and speculating further inferences. Since the retention of autofluorescence is variable, only qualitative information about the chlorophyll content per cell can be derived from the fixed samples (Navaluna et al., 1989). The autofluorescence is influenced by a change in light and nutrients and therefore is an important tool to examine environmentally induced variation in phytoplankton physiology (Veldhuis and Kraay, 2000). Since chlorophyll a is a light sensitive pigment (Ledford and Niyogi, 2005), the natural light protection mechanism does not take place in the fixed samples. This suggests that the fixed samples must be carefully stored and protected against light and temperature fluctuations. In our previous work, even though the cells were in nutrient-rich media, the absence of light triggered the reduction in autofluorescence but the chlorophyll content remained intact (Naik & Anil, unpublished). Therefore, care must be taken when using chlorophyll autofluorescence obtained from FCM as a proxy for chlorophyll. Fixation also has a significant role in the membrane permeability (Troussellier et al., 1995). The cell membrane permeability allows quick and easy penetration of high molecular weight molecules such as nucleic acid fluorochromes (Troussellier et al., 1995). However, fixation and storage method employed should not change the special features (e.g. DNA content) of the cells (Troussellier et al., 1995). In this study, the fixed samples stained with SYBR Green I showed a decrease in green fluorescence compared to the unfixed samples suggesting the permeability of fixed samples was reduced (Fig. 1E). Similar results were also observed in marine bacteria (Kamiya et al., 2007). The results also unveiled that permeability was initially affected by GA concentration but later it was independent. This was reflected in the coefficient of variation (CV) of the samples (data not shown). For FCM analysis, CV is used to represent the spread of data. CV of 1% GA fixed and stained samples were higher compared to lower concentrations of GA until day 1 but eventually GA concentration had no impact. The storage temperatures employed also had insignificant effect on the permeability as indicated by negligible changes in the green fluorescence (Fig. 2E). Thus, these results conclude that the membrane permeability was independent of GA concentration and storage temperature in T. indica.
One common obstacle a phycologist encounters is the cell loss. Cell loss due to fixation and freezing has been previously administered (Marie et al., 2014; Pan et al., 2005; Vaulot et al., 1989). Freezing results in loss of liquid water which increases the concentration of intraand extracellular solutes (Taylor and Fletcher, 1998). This causes lethal stress to the cells and results in cell loss. After an initial cell loss on fixation (16–19%), the cell recovery was best even after 6 months at 5 °C compared to other storage temperatures (− 80 °CLN2, − 80 °C, − 20 °C). More than the concentration of GA, storage temperature had a significant impact on the cell loss of T. indica (Fig. 1A and 2A). Storage at − 80 °C and liquid nitrogen (− 196 °C) is normally recommended for long-term storage of phytoplankton as higher temperature (e.g. − 20 °C) may result in rapid sample degradation (Marie et al., 2005). In contrast, the results from this study showed ~ 41% cell loss after 4 months at −80 °CLN2, − 80 °C, and −20 °C indicating freezing is not suitable for long-term storage of all species. The cell recovery was also maintained satisfactorily in −20 °C until 4 months with minimal degradation thereby suggesting that these differences may be due to the differences in the taxonomic groups. Considering FCM discriminates phytoplankton from other particles based on their cellular parameters (Marie et al., 2005), the fixative and storage temperature employed must preserve these properties of cells. The fixed samples are also used for carbon, biomass, and biovolume estimation (Montagnes et al., 1994). On that account, the cell dimensions after fixation should be close to the actual values. Otherwise, these results can be erroneous and may result in underestimation or overestimation of these parameters. Observation showed a change in the cell size immediately after fixation. The highest increase in the cell size was seen in 1% fixed samples (Fig. 1B). This increase can be due to the change in the refractive index by aldehyde (Cucci and Sieracki, 2001) or cellular swelling during storage (Sato et al., 2006). Although the cell granularity decreased immediately after fixation, later it was independent of GA concentration (Fig. 1C). In addition to fixation, the cell size was also influenced by storage temperature (Fig. 2B). The cell size of − 80 °CLN2 and 5 °C preserved samples were equivalent to the unfixed samples even after 6 months. However, the scatter characteristics of different storage temperatures revealed a different story (Fig. 3). The samples stored at 5 °C evinced homogenous population even after 6 months (Fig. 3D–F) whereas other storage temperatures (− 20 °C, − 80 °C, and − 80 °CLN2) displayed heterogeneity (Fig. 3G–O). Thus indicating that 5 °C storage temperature is an ideal choice to maintain the cell integrity of T. indica. Fixation is not only needed to maintain the cell integrity and minimize the morphological changes but also to retain the autofluorescence. When the autofluorescent properties of the pigments are retained satisfactorily, samples can be preserved for delayed analysis (Hall, 1991). Since the autofluorescence of the unfixed samples can be lost rapidly, fixation of the cells is a must (Hall, 1991). In this study, ~ 50% reduction in the autofluorescence of unfixed samples of T. indica was observed after 6 months when stored at 5 ºC (Fig. 2D). Therefore, the present results recommend the fixation of cells being mandatory, especially for long-term storage. This does not imply that fixation does not affect the autofluorescence. The autofluorescence was greatly impacted due to fixation and storage temperature. A higher concentration of GA (1%) was better at retaining the autofluorescence compared to 0.25% GA when stored at 5 °C (Fig. 1D). In the long run, with 0.25% GA, the autofluorescence was retained adequately well at lower temperatures such as − 80 °C and −80 °CLN2 compared to − 20 °C and 5 °C. This decline in autofluorescence with a lower concentration of GA (0.25%) and higher storage temperature (−20 °C and 5 °C) could be possibly due to decreased absorption of light by pigments and decreased yield in fluorescence re-emission of absorbed light (Navaluna et al., 1989). It could also be the result of deterioration of photosynthetic pigments due to chemical fixation and leakage of pigments through compromised cell membrane during storage (Sato et al., 2006). Therefore, fixation of cells with a lower concentration of GA (0.25%)
5. Conclusions Fixation and storage temperature significantly influenced the cell recovery and cellular parameters. Cell recovery was independent of GA concentration but dependent on storage temperature. Cell size and 127
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Fig. 3. Flow cytometric signatures of Tetraselmis indica for unfixed (control; A–C) samples on day 0 and 0.25% glutaraldehyde fixed samples stored at temperature 5 ºC (D–F), − 20 ºC (GI), −80 ºC (J–L), and − 80 °CLN2 (M–O) for 6 months.
accurate representation of the living cells (well preserved morphology and intact flagella) when observed using light microscopy, GA is recommended as an ideal fixative for microscopic analysis of Tetraselmis species. Even though this is a single species study, the results described here can be used as a starting point for the establishment of a protocol for other phytoplankton species as well as to elucidate the often neglected role of storage temperature.
chlorophyll autofluorescence were dependent on GA concentration and storage temperature while cell granularity and cell membrane permeability were independent of these factors except for − 20 °C stored samples. Since 0.25% GA resulted in poor autofluorescence retention at 5 °C, preservation temperature helped in overcoming this negative influence. Therefore, in addition to fixation, the role of storage temperature in the retention of autofluorescence and other cellular parameters cannot be ruled out and must be optimized for each phytoplankton under study. These findings suggest the use of lower concentration (0.25%) of GA when stored at a lower temperature (− 80 °CLN2 and − 80 °C) while a higher concentration (1%) of GA when stored at a relatively higher temperature (5 °C); particularly to retain the chlorophyll autofluorescence. Since GA maintained the
Acknowledgements This work was supported by Council of Scientific & Industrial Research (CSIR) funded project Ocean Finder (PSC0105). The author S.M.N. is a student of Academy of Scientific and Innovative Research 128
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(AcSIR) and greatly acknowledges the research fellowship awarded by University Grants Commission (UGC). S.M.N. is also grateful to Dr. Lalita Baragi, Dr. Dhiraj Narale, and Mr. Kaushal Mapari for their continuous help. The authors have no conflict of interest with respect to this work. This is the CSIR-NIO contribution 6057. References Arora, M., Anil, A.C., Leliaert, F., Delany, J., Mesbahi, E., 2013. Tetraselmis indica (Chlorodendrophyceae, Chlorophyta), a new species isolated from salt pans in Goa, India. Eur. J. Phycol. 48, 61–78. http://dx.doi.org/10.1080/09670262.2013.768357. Azma, M., Mohamed, M.S., Mohamad, R., Rahim, R.A., Ariff, A.B., 2011. Improvement of medium composition for heterotrophic cultivation of green microalgae, Tetraselmis suecica, using response surface methodology. Biochem. Eng. J. 53, 187–195. http:// dx.doi.org/10.1016/j.bej.2010.10.010. Bloem, J., Bar-Gilissen, M.J.B., Cappenberg, T.E., 1986. Fixation, counting, and manipulation of heterotrophic nanoflagellates. Appl. Environ. Microbiol. 52, 1266–1272. Carney, K.J., Basurko, O.C., Pazouki, K., Marsham, S., Delany, J.E., Desai, D.V., Anil, A.C., Mesbahi, E., 2013. Difficulties in obtaining representative samples for compliance with the ballast water management convention. Mar. Pollut. Bull. 68, 99–105. http:// dx.doi.org/10.1016/j.marpolbul.2012.12.016. Cloern, J.E., Foster, S.Q., Kleckner, A.E., 2014. Phytoplankton primary production in the world's estuarine-coastal ecosystems. Biogeosciences 11, 2477–2501. http://dx.doi. org/10.5194/bg-11-2477-2014. Cucci, T.L., Sieracki, M.E., 2001. Effects of mismatched refractive indices in aquatic flow cytometry. Cytometry 44, 173–178. http://dx.doi.org/10.1002/10970320(20010701)44:3<173::AID-CYTO1109>3.0.CO;2-5. Falkowski, P.G., 1994. The role of phytoplankton photosynthesis in global biogeochemical cycles*. Photosynth. Res. 3, 235–258. http://dx.doi.org/10.1007/BF00014586. Fritzsch, F.S.O., Dusny, C., Frick, O., Schmid, A., 2012. Single-cell analysis in biotechnology, systems biology, and biocatalysis. Annu. Rev. Chem. Biomol. Eng. 3, 129–155. http://dx.doi.org/10.1146/annurev-chembioeng-062011-081056. Guillard, R.R.L., Ryther, J.H., 1962. Studies of marine planktonic diatoms I. Cyclotella nana Hustedt, and Detonula confervasea (Cleve) Gran. Can. J. Microbiol. 8, 229–239. http://dx.doi.org/10.1139/m62-029. Hall, J.A., 1991. Long-term preservation of picophytoplankton for counting by fluorescence microscopy. Br. Phycol. J. 26, 169–174. http://dx.doi.org/10.1080/ 00071619100650131. Hopwood, D., 1967. Some aspects of fixation with glutaraldehyde. A biochemical and histochemical comparison of the effects of formaldehyde and glutaraldehyde fixation on various enzymes and glycogen, with a note on penetration of glutaraldehyde into liver. J. Anat. 101, 83–92. Jones, D., 1976. Chemistry of fixation and preservation with aldehydes. In: Steedman, H.F. (Ed.), Zooplankton Fixation and Preservation. Monographs of Oceanographic Methodology. Unesco Press, Paris, pp. 155–171. http://dx.doi.org/10.1139/f77-187. Kamiya, E., Izumiyama, S., Nishimura, M., Mitchell, J., Kogure, K., Amiya, E., Zumiyama, S., Ishimura, M., Itchell, J., Ogure, K., 2007. Effects of fixation and storage on flow cytometric analysis of marine bacteria. J. Oceanogr. 63, 101–112. http://dx.doi.org/ 10.1007/s10872-007-0008-7. Katano, T., Yoshida, M., Lee, J., Han, M.-S., Hayami, Y., 2009. Fixation of Chattonella antiqua and C. marina (Raphidophyceae) using Hepes-buffered paraformaldehyde and glutaraldehyde for flow cytometry and light microscopy. Phycologia 48, 473–479.
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Long-term preservation of Tetraselmis indica (Chlorodendrophyceae, Chlorophyta) for flow cytometric analysis: Influence of fixative and storage temperature
MARK
Sangeeta Mahableshwar Naik, Arga Chandrashekar Anil⁎ Academy of Scientific and Innovative Research (AcSIR), CSIR-National Institute of Oceanography (NIO), Dona Paula 403 004, Goa, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Tetraselmis indica Flow cytometry Glutaraldehyde Storage temperature Chlorophyll autofluorescence
Immediate enumeration of phytoplankton is seldom possible. Therefore, fixation and subsequent storage are required for delayed analysis. This study investigated the influence of glutaraldehyde (GA) concentrations (0.25%, 0.5%, and 1%) and storage temperatures (−80 °CLN2, −80 °C, −20 °C, and 5 °C) on Tetraselmis indica for flow cytometric analysis. Cell recovery, granularity, and membrane permeability were independent of GA concentration whereas cell size and chlorophyll autofluorescence were concentration dependent. After an initial cell loss (16–19%), no cell loss was observed when samples were stored at 5 °C. Cell recovery was not influenced by storage temperature until 4 months but later samples preserved at −80 °CLN2, −80 °C, and −20 °C resulted in ~41% cell loss. Although maximum cell recovery with minimal effect on cell integrity was obtained at 5 °C, autofluorescence was retained better at − 80 °CLN2 and −80 °C. This suggests that in addition to fixative, the choice of storage temperature is equally important. Thus for long-term preservation, especially to retain autofluorescence, the use of lower concentration (0.25%) of GA when stored at a lower temperature (−80 °CLN2 and − 80 °C) while a higher concentration (1%) of GA when stored at a higher temperature (5 °C) is recommended.
1. Introduction Phytoplankton are the foundation of marine food web dynamics, in spite of their small size, on which relies most of the aquatic life forms. In addition to their role in biogeochemical cycles, they also contribute to 40% of the global primary productivity (Falkowski, 1994). Considering they are important producers in the world's estuarine and coastal waters (Cloern et al., 2014), any shift in their community structure can alter the trophodynamics. Traditionally, phytoplankton were enumerated using microscopy but with the advent of automated technology such as flow cytometry (FCM); cell counting is faster, efficient, precise, and prone to minimal errors. FCM not only enables cell counting and sorting (e.g. Marie et al., 2017), but also facilitates cell cycle analysis and simultaneous measurements of multiple cellular parameters (cell size, cell granularity, autofluorescence, and fluorochrome-induced fluorescence) of an individual cell (Marie et al., 2005). Although phytoplankton studies are recommended in the real time i.e., fresh and unfixed, it is not always a feasible approach. Thus, fixation of samples and subsequent storage is mandatory. The effect of fixation and preservation on phytoplankton has been evaluated in
several studies, both for laboratory cultures and natural population (Katano et al., 2009; Marie et al., 2014; Naik et al., 2010; Pan et al., 2005; Troussellier et al., 1995). However, fixation of fragile organisms such as prasinophytes and cryptomonads engenders significant cell loss and alters cellular characteristics (Murphy and Haugen, 1985). A universal protocol of fixation and storage for all phytoplankton is difficult. The best way to overcome this problem is to optimize the protocol based on the target phytoplankton, aim, and duration of the study. Numerous fixatives are available for phytoplankton studies, yet not all are suitable for FCM analysis. For FCM analysis, the fixative used must allow the cells to remain in single-cell suspension. Single-cell analysis has been recognized as an important technology to elucidate key cellular functions which are otherwise difficult from bulk measurements (Fritzsch et al., 2012). Likewise, the chlorophyll autofluorescence is also a pre-requisite for the distinction between autotrophic and heterotrophic phytoplankton (Bloem et al., 1986; Navaluna et al., 1989). As a result, an ideal fixative should preserve both these properties of the cells. Paraformaldehyde (PFA) and glutaraldehyde (GA) are the most popular and effective fixatives used for FCM analysis of both natural population and laboratory cultures of phytoplankton (e.g. Katano et al., 2009; Marie et al., 2014, 2005; Sato et al., 2006;
Abbreviations: CV, coefficient of variation; FCM, flow cytometry; GA, glutaraldehyde; PBS, phosphate-buffered saline; PFA, paraformaldehyde ⁎ Corresponding author at: CSIR-National Institute of Oceanography, Dona Paula 403 004, Goa, India. E-mail address: [email protected] (A.C. Anil). http://dx.doi.org/10.1016/j.mimet.2017.05.018 Received 25 April 2017; Received in revised form 29 May 2017; Accepted 29 May 2017 Available online 30 May 2017 0167-7012/ © 2017 Elsevier B.V. All rights reserved.
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temperature (n = 3 replicates × 5 sampling days = 15).
Vaulot et al., 1989) because they preserve both cellular structures and pigment autofluorescence. In this study, Tetraselmis indica was used as a model organism. Tetraselmis species were traditionally regarded as the members of Prasinophyceae but recent work has proven their relationship with the core Chlorophyta (Arora et al., 2013; Leliaert et al., 2012). Tetraselmis indica was isolated from a saltpan of Goa where salinity and temperature ranged from 35 to 350 and 28 to 48 °C respectively (Arora et al., 2013). Tetraselmis species even have implications in ballast water management programs (e.g. Carney et al., 2013). Due to such extreme environment of saltpans, this alga has to possess a remarkable physiology to adapt to these conditions. Our recent work on T. indica has revealed their interesting physiology by adapting to 6 months of darkness and retaining the growth potential (Naik & Anil, unpublished). Therefore, to study the physiological adaptation of T. indica to various environmental conditions, a suitable fixation and storage protocol is desirable. Classical fixatives such as Lugol's iodine, formalin, and methanol have been frequently used to fix and preserve Tetraselmis species (e.g. Azma et al., 2011; Carney et al., 2013; Ozbay and Jackson, 2010). The problem with these fixatives is that Lugol's and formalin modify the cell shape and affect the fluorescence whereas alcohol (methanol, ethanol) extracts the lipophilic pigments thereby causing the loss of chlorophyll autofluorescence (Marie et al., 2005). Owing to this, these fixatives cannot be used for FCM analysis. Fixing T. indica with PFA prepared using phosphate-buffered saline (PBS-PFA) resulted in the formation of cell aggregates which failed to meet the first criterion for FCM analysis i.e., single-cell suspension. Therefore, GA was the next fixative of choice. In view of this, the objective of this study was to evaluate the 1) influence of GA concentration on cell recovery and cellular parameters of T. indica and 2) role of storage temperature in long-term preservation of T. indica.
2.3. FCM analysis Sample analysis was performed using FACS Verse flow cytometer (BD Biosciences- US) equipped with a blue laser (488 nm). The data acquisition was triggered on red fluorescence (700/54 nm). The day-today cytometer performance and consistency were maintained and standardized using BD FACSSuite™ CS & T research beads. Forward scatter (a proxy for cell size), side scatter (a proxy for cell granularity), red fluorescence (a proxy for chlorophyll autofluorescence), and green fluorescence (SYBR Green I induced) was recorded in list mode and processed using FACS Suite software. Fluoresbrite® YG microspheres of size 10.3 μm (Polysciences Co., USA) were added as an internal standard to compare the cellular parameters of different samples. The normalization of cellular parameters was done by dividing the median value of cell population with the median value of beads (Marie et al., 2014). The effect of fixative and storage temperature on green fluorescence (a proxy for cell membrane permeability) was analyzed by staining the samples with 10 μM (final concentration) SYBR Green I (Invitrogen) followed by dark incubation for 1 h. For each sample, 10,000 events were recorded. 2.4. Statistical analyses The normality and homogeneity of the data were determined using the Shapiro Wilk's and Levene's test to meet the assumptions of parametric tests. The variation in various parameters with days was carried using one-way analysis of variance and Tukey's post hoc test was applied. All the statistical analyses were performed using the SPSS statistical package (Windows Version 22). 3. Results
2. Material and methods 3.1. Effect of GA concentrations 2.1. Culture conditions After an initial cell loss (16–19%) on fixing (p < 0.001, Tukey's post hoc test) due to cell death or lysis, insignificant cell loss occurred later (p > 0.05, Tukey's post hoc test; Fig. 1A). This indicates that the cell recovery was independent of the GA concentration tested. However, a significant change in cell size, granularity, autofluorescence, and membrane permeability was observed immediately after fixation compared to the unfixed samples of day 0 (15 min) (p < 0.001, Tukey's post hoc test; Fig. 1B–E). The addition of GA increased the cell size but decreased the granularity, autofluorescence, and membrane permeability (p < 0.001, Tukey's post hoc test). The increase in cell size was maximum in 1% (44.88%) fixed samples followed by 0.5% (39.38%) and 0.25% GA (29.68%) (p < 0.001, Tukey's post hoc test). The change in cell granularity was influenced by the GA concentration initially; with insignificant change in 0.25% fixed samples (p > 0.05, Tukey's post hoc test) and maximum change in 1% fixed samples (p < 0.001, Tukey's post hoc test). Later, insignificant variations were observed and it was independent of GA concentrations (p > 0.05, Tukey's post hoc test). At the end of 1 month, the autofluorescence was reduced by 15.13% in 1% fixed samples followed by 0.5% (23.16%) and 0.25% (28.73%). Apart from the initial fluctuations (till day1), the membrane permeability was independent of GA concentrations (p > 0.05, Tukey's post hoc test). Interestingly, the membrane permeability was lower for fixed samples compared to the unfixed samples.
Tetraselmis indica was maintained in f/2 without silicate enriched natural seawater (salinity 35) at 25 °C ± 2 °C (Guillard and Ryther, 1962). The light intensity of 40 μmol photons m− 2 s− 1 was provided by daylight white fluorescent tubes with a photoperiod of 12:12 light:dark cycle. The culture flasks were gently shaken by hand every day to ensure homogenous mixing. 2.2. Experimental design The effect of GA concentration was assessed on the exponentially growing (8-days) culture of T. indica. A control of three individual replicates without fixation was analyzed on day 0 while the remaining samples were stored in dark at 5 °C (n = 3 replicates × 5 sampling days = 15). Samples (1.5 mL) fixed with three concentrations of GA (Sigma; electron microscopy grade; final concentration: 0.25%, 0.5%, and 1%) were incubated in dark for 15 min at room temperature. One triplicate set was analyzed immediately (15 min) after fixation while the other sets were stored in dark at 5 °C (n = 3 replicates × 5 sampling days = 15). After 1 day, 7 days, 13 days, and 1 month; samples were thawed at room temperature before FCM analysis. The percentage of cell recovery was obtained with reference to unfixed samples of day 0 (labelled as control- 15 min). To understand the role of storage temperature in cell recovery and preservation of cellular parameters, fixed (0.25% GA) as well as unfixed samples were stored at four different temperatures: 1) −80 °CLN2, 2) − 80 °C, 3) −20 °C, and 4) 5 °C. In − 80 °CLN2 treatment, eppendorf tubes containing samples were immersed in liquid nitrogen followed by storage at − 80 °C. Samples were thawed and analyzed after 1 day, 1 month, 2 months, 4 months, and 6 months of storage at the respective
3.2. Effect of storage temperatures The cell recovery was independent of GA concentrations tested. As a result, to elucidate the influence of storage temperature on cell recovery and cellular parameters, four storage temperatures were tested. For this purpose, 0.25% GA concentration was used. 124
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Fig. 1. Effect of glutaraldehyde concentrations (0% (control), 0.25%. 0.5% and 1%) on Tetraselmis indica stored at 5 ºC for different time interval. A) Percentage of cell recovery, B) Forward scatter (a proxy for cell size), C) Side scatter (a proxy for cell granularity), D) Red fluorescence (a proxy for chlorophyll autofluorescence), and E) Green fluorescence (a proxy for cell membrane permeability). Percentage of cell recovery was obtained with reference to unfixed samples of day 0 (labelled as control-15 min). The dotted line indicates the normalized value of day 0 unfixed samples prior to fixation and storage. Values are mean ± SD (n = 3).
by 34.6% within 1 day in samples stored at 5 °C. Although it decreased at all storage temperatures tested, the maximum reduction of 41.43% was observed in samples stored at 5 °C for 6 months. The samples stored at − 80 °C and − 80 °CLN2 showed the best autofluorescence retention with only 18–23% reduction after 6 months. The membrane permeability was independent of storage temperatures (p > 0.05, Tukey's post hoc test) except for 5 °C initially and at − 20 °C later (Fig. 2E).
The cell recovery was almost constant at all storage temperatures till 4 months (p > 0.05, Tukey's post hoc test; Fig. 2A). Later, ~ 41% cell loss was observed at − 80 °CLN2, − 80 °C, and − 20 °C storage temperatures (p < 0.001, Tukey's post hoc test) while at 5 °C the change was negligible (p > 0.05, Tukey's post hoc test). After 6 months, the cell size in −80 °CLN2 and 5 °C stored samples was almost same and relatively similar to the unfixed samples (p > 0.05, Tukey's post hoc test) compared to other storage temperatures (Fig. 2B). The cell granularity did not show significant variation at −80 °CLN2, −80 °C, and 5 °C storage temperature (p > 0.05, Tukey's post hoc test) except for − 20 °C (p < 0.001, Tukey's post hoc test; Fig. 2C). The autofluorescence retention was significantly different at all storage temperatures (p < 0.001, Tukey's post hoc test; Fig. 2D) and was reduced
4. Discussion Enumeration of phytoplankton is a tedious process and therefore immediate analysis is seldom possible. Consequently, fixation is indispensable to avoid deterioration of samples for delayed FCM or 125
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Fig. 2. Effect of storage temperatures (− 80 °CLN2, − 80 °C, − 20 °C, and 5 °C) on Tetraselmis indica fixed with 0.25% glutaraldehyde for the different time interval. A) Percentage of cell recovery, B) Forward scatter (a proxy for cell size), C) Side scatter (a proxy for cell granularity), D) Red fluorescence (a proxy for chlorophyll autofluorescence), and E) Green fluorescence (a proxy for cell membrane permeability). Percentage of cell recovery was obtained with reference to unfixed samples of day 0 (labelled as control- 15 min). The dotted line indicates the normalized value of day 0 unfixed samples prior to fixation and storage. Values are mean ± SD (n = 3).
single-cell suspension and chlorophyll autofluorescence retention. Despite PFA being one of the popular choices for FCM analysis, in this study, PBS-PFA resulted in the formation of cell aggregates which was observed microscopically. This could be due to the extrusion of cellular material and enhanced scattering due to PFA (Navaluna et al., 1989). For this reason, PFA was regarded unsuitable for the fixation of T. indica. Precipitation on using PBS-PFA has also been reported in earlier study (Katano et al., 2009). Since GA is stable over time, commercially available, and less toxic than PFA (Marie et al., 2014); it was the next preferred choice. GA is also known to maintain the biomass of preserved plankton (Kimmerer and McKinnon, 1986), easily and rapidly cross-links proteins, and inactivates enzymes during fixation than other aldehydes (Hopwood, 1967).
microscopic analysis. Fixation is also needed to enhance the cell membrane permeability (Navaluna et al., 1989) for molecular biology studies (e.g. immunofluorescence, cell cycle analysis) as well as for microscopic examination of phytoplankton. A fixative can be an ideal choice for one organism but a bad choice to another organism. This is because different phytoplankton cultures react differently to fixation (Marie et al., 2014). An ideal fixative should preserve cells against the microbial activity, osmotic damage, autolysis, and maintain an accurate structure representation of the living cells (Jones, 1976). The objective of this study was to develop a preservation protocol for FCM analysis of T. indica and obtain maximum cell recovery with no or least effect on the cellular parameters. The obligatory attributes for FCM analysis which a fixative must possess are an ability to maintain 126
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when stored at a lower temperature (− 80 °CLN2 and −80 °C) and a higher concentration of GA (1%) when stored at a relatively higher temperature (5 °C) is recommended to retain the autofluorescence. The autofluorescence retention can also depend on the physiological status, taxonomic groups, and thawing (Bloem et al., 1986; Sato et al., 2006; Taylor and Fletcher, 1998). In this study, healthy and exponentially growing cells were used. Therefore, the impact of the chemical fixation or storage temperature due to the physiological status of cells can be ignored. Studies carried out on picoplankton population fixed with 2% GA (Hall, 1991) and heterotrophic- and phototrophicnanoflagellates fixed with 1% GA (Bloem et al., 1986) could not retain the autofluorescence satisfactorily. On the other hand, cultured Chlorella species and Haematococcus pluvialis, as well as green algae and cyanobacteria from field samples demonstrated longer retention of autofluorescence (Bloem et al., 1986). The authors attributed this difference in the autofluorescence retention to be dependent on the type of organism under study. However, they did not comment on the role of storage temperature. Additionally, phytoplankton are subjected to series of chemical and physical stress during freezing, low-temperature storage, and thawing; which may result in loss of cell viability (Taylor and Fletcher, 1998) and eventually autofluorescence. Regardless of the fact that the cell autofluorescence is used as a proxy for chlorophyll autofluorescence, care must be taken before relying on it and speculating further inferences. Since the retention of autofluorescence is variable, only qualitative information about the chlorophyll content per cell can be derived from the fixed samples (Navaluna et al., 1989). The autofluorescence is influenced by a change in light and nutrients and therefore is an important tool to examine environmentally induced variation in phytoplankton physiology (Veldhuis and Kraay, 2000). Since chlorophyll a is a light sensitive pigment (Ledford and Niyogi, 2005), the natural light protection mechanism does not take place in the fixed samples. This suggests that the fixed samples must be carefully stored and protected against light and temperature fluctuations. In our previous work, even though the cells were in nutrient-rich media, the absence of light triggered the reduction in autofluorescence but the chlorophyll content remained intact (Naik & Anil, unpublished). Therefore, care must be taken when using chlorophyll autofluorescence obtained from FCM as a proxy for chlorophyll. Fixation also has a significant role in the membrane permeability (Troussellier et al., 1995). The cell membrane permeability allows quick and easy penetration of high molecular weight molecules such as nucleic acid fluorochromes (Troussellier et al., 1995). However, fixation and storage method employed should not change the special features (e.g. DNA content) of the cells (Troussellier et al., 1995). In this study, the fixed samples stained with SYBR Green I showed a decrease in green fluorescence compared to the unfixed samples suggesting the permeability of fixed samples was reduced (Fig. 1E). Similar results were also observed in marine bacteria (Kamiya et al., 2007). The results also unveiled that permeability was initially affected by GA concentration but later it was independent. This was reflected in the coefficient of variation (CV) of the samples (data not shown). For FCM analysis, CV is used to represent the spread of data. CV of 1% GA fixed and stained samples were higher compared to lower concentrations of GA until day 1 but eventually GA concentration had no impact. The storage temperatures employed also had insignificant effect on the permeability as indicated by negligible changes in the green fluorescence (Fig. 2E). Thus, these results conclude that the membrane permeability was independent of GA concentration and storage temperature in T. indica.
One common obstacle a phycologist encounters is the cell loss. Cell loss due to fixation and freezing has been previously administered (Marie et al., 2014; Pan et al., 2005; Vaulot et al., 1989). Freezing results in loss of liquid water which increases the concentration of intraand extracellular solutes (Taylor and Fletcher, 1998). This causes lethal stress to the cells and results in cell loss. After an initial cell loss on fixation (16–19%), the cell recovery was best even after 6 months at 5 °C compared to other storage temperatures (− 80 °CLN2, − 80 °C, − 20 °C). More than the concentration of GA, storage temperature had a significant impact on the cell loss of T. indica (Fig. 1A and 2A). Storage at − 80 °C and liquid nitrogen (− 196 °C) is normally recommended for long-term storage of phytoplankton as higher temperature (e.g. − 20 °C) may result in rapid sample degradation (Marie et al., 2005). In contrast, the results from this study showed ~ 41% cell loss after 4 months at −80 °CLN2, − 80 °C, and −20 °C indicating freezing is not suitable for long-term storage of all species. The cell recovery was also maintained satisfactorily in −20 °C until 4 months with minimal degradation thereby suggesting that these differences may be due to the differences in the taxonomic groups. Considering FCM discriminates phytoplankton from other particles based on their cellular parameters (Marie et al., 2005), the fixative and storage temperature employed must preserve these properties of cells. The fixed samples are also used for carbon, biomass, and biovolume estimation (Montagnes et al., 1994). On that account, the cell dimensions after fixation should be close to the actual values. Otherwise, these results can be erroneous and may result in underestimation or overestimation of these parameters. Observation showed a change in the cell size immediately after fixation. The highest increase in the cell size was seen in 1% fixed samples (Fig. 1B). This increase can be due to the change in the refractive index by aldehyde (Cucci and Sieracki, 2001) or cellular swelling during storage (Sato et al., 2006). Although the cell granularity decreased immediately after fixation, later it was independent of GA concentration (Fig. 1C). In addition to fixation, the cell size was also influenced by storage temperature (Fig. 2B). The cell size of − 80 °CLN2 and 5 °C preserved samples were equivalent to the unfixed samples even after 6 months. However, the scatter characteristics of different storage temperatures revealed a different story (Fig. 3). The samples stored at 5 °C evinced homogenous population even after 6 months (Fig. 3D–F) whereas other storage temperatures (− 20 °C, − 80 °C, and − 80 °CLN2) displayed heterogeneity (Fig. 3G–O). Thus indicating that 5 °C storage temperature is an ideal choice to maintain the cell integrity of T. indica. Fixation is not only needed to maintain the cell integrity and minimize the morphological changes but also to retain the autofluorescence. When the autofluorescent properties of the pigments are retained satisfactorily, samples can be preserved for delayed analysis (Hall, 1991). Since the autofluorescence of the unfixed samples can be lost rapidly, fixation of the cells is a must (Hall, 1991). In this study, ~ 50% reduction in the autofluorescence of unfixed samples of T. indica was observed after 6 months when stored at 5 ºC (Fig. 2D). Therefore, the present results recommend the fixation of cells being mandatory, especially for long-term storage. This does not imply that fixation does not affect the autofluorescence. The autofluorescence was greatly impacted due to fixation and storage temperature. A higher concentration of GA (1%) was better at retaining the autofluorescence compared to 0.25% GA when stored at 5 °C (Fig. 1D). In the long run, with 0.25% GA, the autofluorescence was retained adequately well at lower temperatures such as − 80 °C and −80 °CLN2 compared to − 20 °C and 5 °C. This decline in autofluorescence with a lower concentration of GA (0.25%) and higher storage temperature (−20 °C and 5 °C) could be possibly due to decreased absorption of light by pigments and decreased yield in fluorescence re-emission of absorbed light (Navaluna et al., 1989). It could also be the result of deterioration of photosynthetic pigments due to chemical fixation and leakage of pigments through compromised cell membrane during storage (Sato et al., 2006). Therefore, fixation of cells with a lower concentration of GA (0.25%)
5. Conclusions Fixation and storage temperature significantly influenced the cell recovery and cellular parameters. Cell recovery was independent of GA concentration but dependent on storage temperature. Cell size and 127
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Fig. 3. Flow cytometric signatures of Tetraselmis indica for unfixed (control; A–C) samples on day 0 and 0.25% glutaraldehyde fixed samples stored at temperature 5 ºC (D–F), − 20 ºC (GI), −80 ºC (J–L), and − 80 °CLN2 (M–O) for 6 months.
accurate representation of the living cells (well preserved morphology and intact flagella) when observed using light microscopy, GA is recommended as an ideal fixative for microscopic analysis of Tetraselmis species. Even though this is a single species study, the results described here can be used as a starting point for the establishment of a protocol for other phytoplankton species as well as to elucidate the often neglected role of storage temperature.
chlorophyll autofluorescence were dependent on GA concentration and storage temperature while cell granularity and cell membrane permeability were independent of these factors except for − 20 °C stored samples. Since 0.25% GA resulted in poor autofluorescence retention at 5 °C, preservation temperature helped in overcoming this negative influence. Therefore, in addition to fixation, the role of storage temperature in the retention of autofluorescence and other cellular parameters cannot be ruled out and must be optimized for each phytoplankton under study. These findings suggest the use of lower concentration (0.25%) of GA when stored at a lower temperature (− 80 °CLN2 and − 80 °C) while a higher concentration (1%) of GA when stored at a relatively higher temperature (5 °C); particularly to retain the chlorophyll autofluorescence. Since GA maintained the
Acknowledgements This work was supported by Council of Scientific & Industrial Research (CSIR) funded project Ocean Finder (PSC0105). The author S.M.N. is a student of Academy of Scientific and Innovative Research 128
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