Flow cytometric counts of the picoeukaryote, Micromonas pusilla and 2 um green fluorescent bead abundance (Ochromonas danica was used as the predator) in laboratory-culture experiments to demonstrate the saturation approach.
The experiments used the mixotrophic chrysophyte Ochromonas danica, ~ 6 to 9 µm in diameter, as a predator, and the generally abundant picoeukaryote Micromonas pusilla, ~ 1 to 2 µm in diameter, as prey.
For detailed methods, refer to Archer et al. (2022) in the section 'Tests Using Single Prey and Predator Combinations'.
Culture experiment
The cultures were maintained in sterile L1 media in a 21˚C incubator with a 14 h light/10 h dark cycle and light levels of ~ 90 µE m-2 sec-1. Prior to the start of the experiments, O. danica was transitioned from K media and rice to sterile L1 media and then fed every other day on M. pusilla. The prey, M. pusilla was maintained in semi-continuous growth through regular transfers (2 - 3 days). Similar ratios of predator-to-prey were generated in tubes containing a total volume of 40 ml. Fluorescent polystyrene microspheres (beads) of 2 µm in diameter (Fluoresbrite Plain YG microspheres, Polysciences, Inc., Warrington, PA), were used as surrogate prey. To minimize clumping, the beads were blocked in a solution of 1% bovine serum albumin overnight then centrifuged for 5 minutes at 2000 rpm, after which the pellet was resuspended in 0.2 µm filtered seawater. A new solution of beads was prepared at the start of each experiment. During the incubations, the experimental tubes were rotated at 1.2 rpm on a plankton wheel to keep particles in suspension. Two subsamples of 1 ml were removed from each tube at the start (T0) and the final time point (T24) after ~24 hours, for flow cytometric analysis.
Flow Cytometric Measurements
Particles were excited with a 488 nm blue excitation laser (100 mW). Data acquisition was triggered on forward scatter (FSC). Signals were recorded from detectors with bandpass filters for right angle light scatter and fluorescence emission in red (692 nm/80 nm band pass) indicative of chlorophyll a, orange for phycoerythrin (593/52 nm), and green (525/35 nm). To ensure accurate calibration of the flow cytometer, ZE5 QC beads (Bio-Rad, Hercules, CA, USA) were run daily.
Archer, S. D., Poulton, N. J. (2023) Flow cytometric counts from grazing saturation culture experiment using single prey (Micromonas pusilla) and predator (Ochromonas danica) in October 2019. Biological and Chemical Oceanography Data Management Office (BCO-DMO). (Version 1) Version Date 2023-08-02 [if applicable, indicate subset used]. http://lod.bco-dmo.org/id/dataset/905469 [access date]
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