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Periodicity of Sampling:
Samples are collected on a monthly basis from the R/V Sea-Watch (Southern California Marine Institute) in conjunction with the Wrigley Institute for Environmental Studies at the San Pedro Oceanographic Time Series (SPOTS), located midway between the Port of Los Angeles and Santa Catalina Island (33° 33’ N, 118° 24’ W) in approximately 900 meters of water.
Sampling Depths:
Routine samples are collected with a CTD/Niskin water-sampling rosette from 5 meters, the Chlorophyll a maximum layer (determined by in situ fluorometry), 150 meters, 500 meters and more recently ~880 meters (approximately 10 meters above the seafloor).
Protistan samples:
Microplankton – Preserve a 500 ml water sample collected directly from Niskin bottle to a final concentration of ~5% Lugol’s solution (500 ml sample + 25 ml Lugol’s). Store sample at room temperature until processed.
Heterotrophic nanoplankton – Preserve a 250 ml water sample collected directly from Niskin bottle to a final relative concentration of ~1% formalin (250 ml + 2.5 ml of 0.2 ?m filtered undiluted formalin). Note: Undiluted formalin is ~37%, so the absolute final concentration of formalin in a preserved sample is ~0.37%. Refrigerate sample in the dark until processed.
Protistan DNA – Pre-filter seawater sample with 200 and/or 80 ?m Nitex mesh directly from Niskin bottle (gravity filtration) to remove most metazoa followed by collection of filtrate into acid-washed cubitainers. Filter sub-samples (2 liters each) onto 47 mm GF/F filters by gentle vacuum filtration (<10 mm Hg) to collect protistan biomass. Roll the GF/F filter with filter forceps and transfer individual samples to a 15 ml Falcon tube. Add two ml of cell lysis/storage buffer to the tube containing the filter, making sure to coat the filter with the buffer. Flash-freeze the sample in a liquid nitrogen dry shipper at sea and transfer the sample to -80°C freezer upon return to the lab.
Flow Cytometry – Collect 12 ml samples (3 per depth) from the Nitex-filtered seawater into 15 ml Falcon tubes and preserve with 120 µl of 0.2 µm filtered undiluted formalin (final relative concentration ~1%) before flash-freezing in a liquid nitrogen dry shipper.
Culture enrichments – Collect two 200 ml samples from each depth for phototrophic and heterotrophic protistan enrichment. Add 0.7 ml of f/2 nutrients (0.2 ml NO3, 0.2 ml PO4, 0.2 ml trace metals, 0.1 ml vitamins) to one sample (phototrophs) and two drops of 10% yeast extract plus 2 grains of sterile rice to another sample (heterotrophs).
Plankton Tow – Tow a 10-µm mesh plankton net at the surface and/or down to the chlorophyll a maximum layer (vertical tow) to provide a live sample for single-cell isolation and/or microscopy.
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