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 th...(23)

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.

Data 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.