Abstract:
Collection and preservation of open ocean water samples from stations along a transect in the Barents Sea over the course of a year from July 2017 - July 2018. Four cruises in total to cover seasonal changes, two on board the James Clark Ross (RRS) and two aboard the Helmer Hansen (RV). A standard CTD cast was deployed to collect the samples, the depths were selected to support Primary Production experiments on board the ship, with deep samples representing 1% PAR. Research assistants from SAMS (Scottish Association for Marine Science) were responsible for the sample collection and Elaine Mitchell of SAMS was responsible for the sample analysis and data processing.
This work was funded by Arctic PRIZE - NERC Thematic grant - Changing Arctic Ocean (CAO) programme - NE/P006302/1.
Keywords:
Bacteria, Barents Sea, Biomass, Flow cytometry, Pico-plankton
Mitchell, E., McNeil, S., Whyte, C., Cottier, F., Hopkins, J., & Davidson, K. (2021). Flow cytometry analysis of water samples for bacterial and Pico-plankton enumeration, samples collected in the Barents Sea during 2017-2018 (Version 1.0) [Data set]. UK Polar Data Centre, Natural Environment Research Council, UK Research & Innovation. https://doi.org/10.5285/2182fd8d-0a0d-4f56-9bf9-4dffaa67ff4b
| Access Constraints: | There are no restrictions in accessing the data. |
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| Use Constraints: | Dataset is released under Open Government Licence V3.0: http://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/. |
| Creation Date: | 2020-02-10 |
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| Dataset Progress: | In Work |
| Dataset Language: | English |
| ISO Topic Categories: |
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| Parameters: |
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| Personnel: | |
| Name | UK Polar Data Centre |
| Role(s) | Metadata Author |
| Organisation | British Antarctic Survey |
| Name | Dr Elaine Mitchell |
| Role(s) | Investigator |
| Organisation | Scottish Association for Marine Science |
| Name | Mrs Sharon McNeil |
| Role(s) | Investigator |
| Organisation | Scottish Marine Institute |
| Name | Dr Callum Whyte |
| Role(s) | Investigator |
| Organisation | Scottish Marine Institute |
| Name | Dr Joanne Hopkins |
| Role(s) | Investigator |
| Organisation | NOC |
| Name | Prof Keith Davidson |
| Role(s) | Investigator |
| Organisation | Scottish Association for Marine Science |
| Name | Prof Finlo Cottier |
| Role(s) | Investigator |
| Organisation | Scottish Association for Marine Science |
| Parent Dataset: | N/A |
| Quality: | Setting up of the gates for the three sets of cell analysis was done by hand for each sample run by Elaine Mitchell. Samples for flow cytometry really should be done fresh without freezing, as freezing does cause damage to the cells which in turn can create indistinct dot plots. Gating these plots is tricky but by analysing the samples using a range of plots with different parameters and logic gates this should have reduced any errors to a minimum. Mike Zubkov at SAMS checked the data for errors/alterations before submission. |
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| Lineage/Methodology: | Sample collection: 4 cruises - JR16-006 / HH180101 / HH230418 / JR17-006 Seawater was collected from six depths from a standard environmental CTD cast as close to midday as possible. The CTD was positioned in full sunlight and not in the shadow of the ship. Sampling depths were selected based on the PAR irradiance readings from the CTD at the surface of the water (approx. 2m) after being initially stabilised at 10m and bought back to the surface.Set percentages of light 100%, 50%, 25%,15%,3% and 1% were calculated from the surface PAR and the depths chosen accordingly. For flow cytometry samples three depths were selected - Surface, Chlorophyll maximum and deep (exception was on HH180101 where only surface waters were taken due to it being wintertime). 10L acid washed carboys and acid washed tubing with 200µm mesh to pre-screen the water were used to collect the water samples. The carboys were stored in black bags either in the cold room or on deck in a low light area. Location of the collected water for storage until processing was dependent on the temperature of the surface water at the point of collection. 180µl of Glutaraldehyde 25% solution, 1% (final concentration) was dispensed into 5ml Cryovials and then 4ml of water sample was added. Samples were mixed and stored in a fridge at 4-5°C for a minimum of 4 hours to allow the fixative to penetrate the cells. After this the samples were dropped into liquid nitrogen to flash freeze them and transferred to -80°C freezer where they remained until analysis. Laboratory analysis: Samples were removed from -80°C freezer in small batches, one or two stations at a time and allowed to defrost in the dark in a refrigerated cool box set at 4°C. Whilst waiting for the samples to defrost the SYBR green Nucleic acid I stain and Citrate buffer were prepared. Citrate buffer: dissolve 4.6g of Potassium Citrate in 50ml of de-ionised water and filter though a sterile 0.2um filter into a sterile 50ml falcon tube - use 100µl/ml. SYBR Green: Remove a micro-centrifuge tube containing a 10µl aliquot of SYBR green from the freezer and defrost. Using a pipette and sterile tip add 250µl of sterile 0.2µm filtered de-ionised water to the micro-centrifuge tube and mix. Transfer this volume to a micro-centrifuge tube fitted with a 0.2µm filter and centrifuge at 13,000rpm for 10 seconds. Remove the tube, add another 250µl of sterile 0.2µm filtered de-ionised water to the filter and re-spin as before. Remove the filter and then transfer the 510µl of SYBR green stock to a new sterile micro-centrifuge tube - use 20µl/ml. Label PE tubes with sample information, to each tube add 300µl of citrate buffer and 60µl of SYBR green stain. Add 3ml of sample to each tube, cover tube with parafilm and leave for a minimum of 30min for the stain to take. Samples were delivered to the FACSort flowcytometer sample probe using BD Discardit II 5ml sterile syringe (non rubber) and a syringe pump that was pre calibrated for set flow rates. Transfer stained samples to a sterile syringe, attach syringe to the FACSort machine via tubing and needles and set up the syringe in the syringe pump cradle to deliver the sample to the flow cytometer for analysis at set rates for set amounts of time. For bacteria samples were ran at 37µl/min for 1 minute, for Pico-plankton samples were ran at 92µl/min for 5 min, total events per second were kept to a maximum of 1000 events per second, but as close to 300-500 as possible to improve accuracy of cell counting. Sample runs were duplicated. Cell quest software was used to analyse each sample using dot plots. Three dot plots set up, FL1 vs SSC for bacteria cells with a threshold set at 20 for FL1. FL1 vs FL2 to help gate out bacteria from the remaining Pico plankton population (no counting on this plot, just for reference). FL1 vs FL3 for pico plankton cells with a threshold set at 150 for FL1. Areas were gated on each plot for analysis, for the bacteria plot there were three gates - Total bacteria, High DNA and Low DNA. For the pico-plankton plot there were 6 gates - Total PNAN, small PNAN, Large PNAN, Total HNAN, small HNAN and large HNAN (PNAN - Phototrophic nanoflagellates, HNAN - Heterotrophic nanoflagellates). Data from each run was displayed in a 'Region statistic' box on Cell Quest and numbers noted into a work book and transferred to an Excel worksheet. The worksheet takes into account the dilution factor from all the additions of fixatives, buffers and stains. The duplicated counts were averaged to provide mean cell numbers/ml. Cell enumeration results for each cell grouping at each station and depth are provided as - mean cell numbers/ml. Bacteria cell sizes are approx. 1-2µm Pico plankton small are approx.. 3-6µm Pico plankton large are approx.. 7-10µm Biomass calculations for bacteria use the cell numbers per ml calculated up to 1 Litre and multiplied by an average carbon value based on Kawasaki et al 2011. Results are provided in pgC/litre for each station at each depth and Total carbon values for the chlorophyll maximum samples are provided in µgC/litre. Biomass calculations for HNAN & PNAN use the cell numbers per ml calculated up to 1 Litre and multiplied by an average carbon value based on cell carbon values from previous Arctic sample analysis from cruises; JR210 & JR219 as the size groupings are the same. Results are provided in pgC/litre for each station at each depth and Total carbon values for the chlorophyll maximum samples are provided in µgC/litre. |
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| Temporal Coverage: | |
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| Start Date | 2018-06-12 |
| End Date | 2018-07-03 |
| Start Date | 2018-04-26 |
| End Date | 2018-05-02 |
| Start Date | 2017-07-08 |
| End Date | 2017-08-05 |
| Start Date | 2018-01-08 |
| End Date | 2018-01-16 |
| Spatial Coverage: | |
| Latitude | |
| Southernmost | 75.3232 |
| Northernmost | 78.1072 |
| Longitude | |
| Westernmost | 15.4823 |
| Easternmost | 30.0508 |
| Altitude | |
| Min Altitude | N/A |
| Max Altitude | N/A |
| Depth | |
| Min Depth | N/A |
| Max Depth | N/A |
| Latitude | |
| Southernmost | 74.8842 |
| Northernmost | 82.5907 |
| Longitude | |
| Westernmost | 9.4731 |
| Easternmost | 31.3314 |
| Altitude | |
| Min Altitude | N/A |
| Max Altitude | N/A |
| Depth | |
| Min Depth | N/A |
| Max Depth | N/A |
| Latitude | |
| Southernmost | 70.7667 |
| Northernmost | 81.5637 |
| Longitude | |
| Westernmost | 10.667 |
| Easternmost | 30.287 |
| Altitude | |
| Min Altitude | N/A |
| Max Altitude | N/A |
| Depth | |
| Min Depth | N/A |
| Max Depth | N/A |
| Latitude | |
| Southernmost | 74.61323 |
| Northernmost | 76.49189 |
| Longitude | |
| Westernmost | 16.76274 |
| Easternmost | 30.04377 |
| Altitude | |
| Min Altitude | N/A |
| Max Altitude | N/A |
| Depth | |
| Min Depth | N/A |
| Max Depth | N/A |
| Location: | |
| Location | Barents Sea |
| Detailed Location | Nansen Basin |
| Location | Barents Sea |
| Detailed Location | Kvitoya |
| Location | Barents Sea |
| Detailed Location | Persey Bank |
| Location | Barents Sea |
| Detailed Location | Norwegian coast - Tromso and Norwegian basin |
| Location | Barents Sea |
| Detailed Location | East of Hoppen Bank |
| Location | Barents Sea |
| Detailed Location | Nordaustlandet |
| Location | Barents Sea |
| Detailed Location | Bear Island |
| Location | Barents Sea |
| Detailed Location | Storfjordrena |
| Location | Barents Sea |
| Detailed Location | Kong Karls Land |
| Location | Barents Sea |
| Detailed Location | Spitzbergen Bank and Spitzbergen shelf edge |
| Data Collection: | Standard CTD and associated software used to determine ocean parameters and define collection depths on board the ship. Sample analysis conducted on a Becton Dickinson FACSort Flow cytometer by Elaine Mitchell. Cell quest software was used to plot the events allowing categorisation of the cells into the three groups. Data from flow cytometry analysis was entered into Comma Separated Value (csv) files for cell enumerations, taking into account dilution factors and the flow rates used. |
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| Data Storage: | There is 12 csv files in total, 3 files for each of the cruises with biomass, counts and summary. |
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