TITLE: Culturing Euplotes crassus to high densities using a combination of algae and bacteria as the food source.
AUTHORS: Lawrence A. Klobutcher, Larry Klobutcher

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">This procedure was developed to investigate the possible use of RNAi through bacterial feeding as a means of knocking down expression of particular genes.</li><li style = "counter-reset:ol0;list-style-type:disc;"><span>While the utility of RNAi is still being investigated, the method is effective in growing </span><span style = "font-style:italic;">Euplotes crassus</span><span> to higher densities than is possible using algae alone. Using moderately dense cultures of algae, we typically achieve cell densities of 500-1,000 cells/ml, while with the E. coli supplemented cultures we describe here, </span><span style = "font-style:italic;">Euplotes</span><span> cell densities exceed 3,000 cells/ml. In fact, preliminary studies mixing a volume of a </span><span style = "font-style:italic;">Euplotes</span><span> culture with an equal volume of resuspended E. coli have produced a density of ~8,000 cells/ml.</span></li><li style = "counter-reset:ol0;list-style-type:disc;">While the described procedure involves small cultures, it can likely be scaled up as required.</li></ul></div><div class = "text-block"><span>For some species of </span><span style = "font-style:italic;">Euplotes</span><span>, long-term culture using bacteria as a food source has not been successful (C. Miceli, personal communication), suggesting that some essential nutrient is not being provided in sufficient quantitities</span></div></div>

[STEPS]
?. Grow a 10 ml culture of E. coli overnight using L-broth. (Notes: we have used strain HT115, but any strain of E. coli will likely do. Do not use antibiotics.)
?. Distribute the bacterial culture to 2 ml microcentrifuge tubes and pellet by centrifugation in a microcentrifuge for 1.5 min.
?. Pour off the supernatant and resuspend the pellet in each tube in 2 ml of a moderately dense culture of Dunaliella salina grown in artificial seawater.
?. In a 15 ml disposable plastic tube, combine 1 ml of a lightly starved Euplotes crassus culture, 7 ml of a moderately dense culture of the algae Dunaliella salina grown on artificial seawater, and 2 ml of the resuspended E. coli culture.
?. Incubate at room temperature with the tubes at an angle.
?. Gently invert tubes daily to disperse settled material.
?. The Euplotes will likely consume all bacteria and algae after about 5 days and should achieve a density of ~3,000 cells/ml.Notes: This procedure was developed to investigate the possible use of RNAi through bacterial feeding as a means of knocking down expression of particular genes.While the utility of RNAi is still being investigated, the method is effective in growing Euplotes crassus to higher densities than is possible using algae alone. Using moderately dense cultures of algae, we typically achieve cell densities of 500-1,000 cells/ml, while with the E. coli supplemented cultures we describe here, Euplotes cell densities exceed 3,000 cells/ml. In fact, preliminary studies mixing a volume of a Euplotes culture with an equal volume of resuspended E. coli have produced a density of ~8,000 cells/ml.While the described procedure involves small cultures, it can likely be scaled up as required.For some species of Euplotes, long-term culture using bacteria as a food source has not been successful (C. Miceli, personal communication), suggesting that some essential nutrient is not being provided in sufficient quantities.