DOI: 10.5285/6e58cace-ea68-4103-8ce4-96a1afeb4835
title: Bacterial and nano-flagellate enumeration and biomass calculations from samples collected in the Greenland Sea across the Fram Strait to Svalbard during May 2018

description: Collection and preservation of open ocean water samples from stations along a transect up the east coastline of Greenland and then across the Fram Strait to Svalbard during May 2018. The cruise was to observe spring bloom conditions, on board the RRS James Clark Ross. A standard CTD cast was deployed to collect the samples, depths were surface, the chlorophyll maximum and a deep sample, selected to support zooplankton net sampling and other on-board experiments. Research assistants from SAMS (Scottish Association for Marine Science) were responsible for the sample collection on JR17005, Elaine Mitchell of SAMS was responsible for the sample analysis and data processing.

Funding was provided from the DIAPOD - NERC thematic grant - Changing Arctic Ocean programme - NE/P006280/1.
description: Seawater was collected from three depths from a standard environmental CTD cast as close to net collections. 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. For bacteria and nano-flagellate samples three depths were selected: surface, the Chlorophyll maximum and a deep sample at approx. 150m. 10L acid washed carboys and acid washed tubing were used to collect the water samples. The carboys were stored in either 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 25% Glutaraldehyde solution, 1% (final concentration) was dispensed into 5ml cryovial tubes. 4ml of water sample was added, the vials were gently inverted and stored in a fridge at 5°C for a minimum of 4 hours (overnight if possible) for the fixative to penetrate the cells before being snap frozen in a dewer flask containing liquid nitrogen. Once frozen the samples were stored in boxes at -80°C 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 through 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 9.5µl/min for 1 minute, for Pico-plankton samples were ran at 95µl/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 workbook and transferred to an Excel worksheet.

The worksheet considers 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 and 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 and 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.
description: 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 Windows 10 Excel spreadsheets for cell enumerations, considering dilution factors and the flow rates used.
description: 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.