Polyethylene caps

Non-axenic clonal cultures were established by isolation of Pseudo-nitzschia cells using drawn out glass pipettes (micropipettes) and a Nikon Eclipse TS100 inverted microscope (≤ 400x magnification) and transferred into 24 multi-well culture plates (Corning Inc. Durham, USA) containing 1 mL f/2 medium [39 ]. These well plates were kept at 16°C– 18°C under a photon flux of 60–100 μmol photon m^-2 s^-1 on a 12/12 hour dark/light cycle (white fluorescent tubes) and checked every alternate day. After 1 week, viable cultures were transferred to 70 mL gamma sterile polystyrene containers with polyethylene caps (Thermo Fisher Scientific, Australia, Pty.) and maintained in the same conditions. One milliliter of culture from each strain was transferred into fresh media every two weeks to establish healthy and exponentially growing monocultures over the duration of the study. On day 14 (late stationary phase) Pseudo-nitzschia cells were harvested for light (LM) and transmission electron microscopy (TEM) examination, DNA sequencing based on the large subunit (LSU) and internal transcribed spacer (ITS1-5.8S-ITS2) regions of the ribosomal DNA, and toxicity determination by liquid chromatography–mass spectrometry (LC-MS/MS) for the presence of DA.

Two nonaxenic monoclonal cultures (TFB_C/18 and TFB_G/18) were established from a net haul sample collected on 2nd August 2018. Single cell isolation of a species of Alexandrium was performed using drawn out glass pipettes (Pasteur pipettes) and a Nikon Eclipse TS100 inverted microscope (100× magnification). Isolated cells were transferred into Falcon^®24 well culture plates containing 1 mL of five-times diluted K medium [29 (link)] without sodium silicate. Germanium dioxide was added at a concentration of 5 µg/mL to prevent diatom growth. Well plates were kept at 18 °C under a photon flux of 60–100 μmol photon m⁻² s ⁻¹ on a 12/12 h dark/light cycle (white fluorescent tubes) and checked every alternate day. After three weeks, monoclonal cultures were transferred into 20 mL of K medium in a 70 mL gamma sterile polystyrene container with polyethylene caps (Thermo Fisher Scientific, Australia, Pty., Scoresby, Vic, Australia) and grown under the same conditions outlined above. One millilitre of culture from each strain was transferred into fresh medium every three weeks to maintain healthy growing cultures.