Diatom cell counts and concentrations during fluorophore PDMPO incubation

To ensure balanced growth and stable conditions, all cultures were acclimated in semi-continuous batch cultures for at least nine generations before starting the experiments. Following acclimation, the experimental incubations were carried out in two consecutive phases: 1) To ensure that cells were acclimated and growing exponentially, cultures were transferred into new media at the end of the exponential growth phase for another nine generations. 2) Following acclimation, cultures were diluted into 8 or 12 incubations bottles (replicate incubations) containing new media. In the first days after transfer, cell concentrations were monitored to ensure exponential growth ("Control phase"). After this short period, [2-(4-pyridyl)-5{[4-dimethylaminoethyl-aminocarbamoyl)-methoxy]phenyl}oxazole] (PDMPO, LysoSensor Yellow/Blue DND-160, Thermo Fisher Scientific, Waltham, MA, USA) was added to four or eight (depending on species and experiment) replicate bottles while the remaining four bottles were incubated without stain addition (controls). Experiments were terminated and sampled 24 h (full light-dark cycle) after PDMPO addition. Daily samples for cell enumeration, fixed with acidic Lugol's solution (around 1% f.c.), were taken during the control phase. After addition of PDMPO, samples for cell enumeration and PDMPO analysis were taken at the time of stain addition (t0) and 24 hours later (t24). Further samples were taken at the beginning (if different from t0) and end (if different from t24) of both light and dark cycles, respectively. Samples were fixed with 2% (f.c.) hexamine-buffered formalin and stored in glass vials in the dark at 4°C until analysis. To determine cell abundance, 10 mL of undiluted or diluted fixed sample (from 3 to 4 independent replicate bottles each) were settled in a 10 ml Utermöhl sedimentation chamber (HYDRO-BIOS, Kiel, Germany). At least 300 cells were counted with a Zeiss Axiovert 40C inverted light microscope or a Zeiss Axiovert 200 epifluorescence microscope. Samples with PDMPO were counted with the aforementioned epifluorescence microscope equipped with a long pass filter (Zeiss Filter set 02; ex: G365, bs: FT395; em: LP420). Samples were counted at 200x to 630x magnification depending on species and intensity of the PDMPO signal.