Culturing Choanos

From ChoanoWiki

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

There are two monoxenic cell lines of the choanoflagellate Monosiga brevicollis. The only difference between the two should be the bacterium with which the choanoflagellates are cultured (the bacteria provides a food source for the choanos). MX1 is cultured with the bacterium Flavobacter, and MX2 is cultured with the bacterium E. aerogenes. There is one monoxenic cell line of Proterospongia sp., PX1, growing in Algoriphagus ALPR1.

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

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