TITLE: Lysozyme-based removal of bacteria from cultures of the marine heterotrophic flagellate Cafeteria roenbergensis
AUTHORS: Monica Berjon-Otero, Sarah Duponchel, Matthias Fischer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol allows to remove bacteria from cultures of the marine heterotrophic nanoflagellate Cafeteria roenbergensis without affecting flagellate viability. Bacterial removal is essential to reduce contamination  and background signal in subsequent microscopy analysis, protein extraction, DNA extraction or other applications. With some changes, this protocol could be adapted to different types of protists.</div></div>

[STEPS]
?. [Preparation of Cafeteria roenbergensis culture]
Determine the cell density of Cafeteria roenbergensis : stain 10 µL of a Cafeteria culture with 1 µL of Lugol’s acid iodine solution and count the cells on a haemocytometer (Neubauer Chamber)
?. [Preparation of Cafeteria roenbergensis culture]
Dilute the Cafeteria culture to 5 x 105 cells/mL with f/2 medium + 0.03% (w/v) yeast extract.Incubate overnight at 22-25 °C.
?. [Preparation of Cafeteria roenbergensis culture]
Determine the flagellate concentration and use approx. 8 x 107 Cafeteria cells/sample
?. [Preparation of Cafeteria roenbergensis culture]
Centrifuge flagellates at 4,500 g, 5 min, room temperature in 50 mL Falcon tubes. Flagellates will be in the pellet.
?. [Preparation of Cafeteria roenbergensis culture]
Resuspend the cell pellets in 1.8 mL of 1X PBS and transfer them into 2 mL microfuge tubes.
?. [Lysozyme and DNaseI treatment]
Add 40 μL of 50 mg/mL freshly prepared lysozyme in 1X PBS (1 mg/mL final concentration).Incubate for 1 hour at room temperature.
Optionally, you can also add 5 mM EDTA here. EDTA increases the activity of lysozyme against Gram negative bacteria, but also negatively affects Cafeteria mobility.
?. [Lysozyme and DNaseI treatment]
Add 200 µL of 10 mg/mL freshly prepared DNaseI in 1X PBS (1 mg/mL final concentration, 2 U/µL) and 11 µL of 1M MgCl2 (5 mM final concentration).Incubate for 30 min at 34°C in a thermomixer with shaking.
?. [Lysozyme and DNaseI treatment]
Centrifuge at 5,000 g, 6 min, 20°C
?. [Optiprep purification]
Resuspend the pellets in 100 µL of 1X gradient buffer (0.5 M NaCl in 1X PBS). Pipette up and down several times to break up aggregates.
?. [Optiprep purification]
Up to 1 x 109 Cafeteria cells can be loaded per SW28 centrifuge tube.Add 1X gradient buffer up to 0.5 - 1 mL final resuspended cell volume.
?. [Optiprep purification]
Load the sample on top of a two-step Optiprep cushion cushion, 20% (w/v) Optiprep at the bottom of the tube and 10% (w/v) Optiprep on top of the 20% layer (both Optiprep solutions are prepared in 1X  gradient buffer).
?. [Optiprep purification]
Centrifuge the tubes in an ultracentrifuge, Beckman SW28 rotor, at 20,000 g, 20 min, 20°C, with slow braking.
?. [Optiprep purification]
Recover Cafeteria cells from the gradient, at the interphase between the 10% and 20% Optiprep layers.
?. [Recovery of Cafeteria culture]
Dilute the recovered Cafeteria cells at least twofold with F/2 medium (no yeast extract)
?. [Recovery of Cafeteria culture]
Diluite the culture 25fold or 50fold  with F/2 and stain 10 µL of this dilution with 1 µL of Lugol’s acid iodine solutionLoad the sample on a haemocytometer to quantify Cafeteria cells and remaining bacteria.
?. [Recovery of Cafeteria culture]
Dilute Cafeteria cells to the desired density. If no or little dilution is required, you can centrifuge again for 6 min at 5,000 g, 20°C and resuspend the cells in F/2 to remove all the remaining Optiprep.