We work with the choanoflagellates in a sterile hood and culture them in a 25°C incubator. If you don’t have an incubator at this temperature, room temperature is fine. We split the cultures every 2-3 days at a dilution of 1:15.

Materials:

Plates (Falcon 35-3003)

Scrapers (Fisher 08-773-1)

Filter-tip pipette tips

Sterile rice (autoclaved in a test tube)

Choano media (seawater infused with Wards Cereal Grass)

To split the cultures:

Scrape the bottom of the dish to resuspend any adherent cells.

Gently pipette up and down with a filter-tip to break up clumps of cells.

Inoculate fresh plate with desired dilution of resuspended plate (we usually split 1:15).

Example of 1:15 inoculation:

14 ml natural choano media (seawater infused with wheatgrass)

1 grain sterile rice (provides a starch source for the bacteria)

1ml choano culture

Stephen Fairclough, adapted from Andrea Morris 2/16/07