Media Materials

• 4L Erlenmeyer Flask
• 4L vaccum Flask (side-arm flask)
• Natural Seawater 2L (can be substitued with synthetic seawater mix made with Milli-Q water.)
• Ward’s cereal grass (Scholar Chemistry #9448606)
• Buchner Funnel
• #1 Whatman (cat# 1001 150)
• 0.22-micron sterile filters (perferably Millipore Steritop/cup)

Choanoflagellate Growth Media

• Boil 2L natural seawater (we usually do this in an autoclave for 20min)
• Add 10g of Ward’s cereal grass to boiling sea water (Scholar Chemistry #9448606)
• Allow to steep until cool (~3-5hrs or overnight is ok)
• The macroscopic cereal grass particles are removed by vacuum filtering twice through Buchner (ceramic) funnel with a double layer of # 1 Whatman (cat# 1001 150)
• This filtered media is sterile filtered through a .22-micron filter in a laminar-flow hood.

Notes

After testing a variety of filtering devices we have found that Millipore Steritops/Stericups filter the fastest and filter more before getting clogged.

The oils from the cereal grass will precipitate with time; allowing longer cooling prior to filtering helps reduce this phenomenon. Also, the media is readily contaminated; so aseptic technique is crucial. We find the using Millipore Stericups is better than using Steritops into glass bottles. With both the Stericup and Steritop systems, the 500mL and 1000mL filters become clogged after about 750mL. If you do use glass bottles, you should autoclave them immediately prior to filtering to use. Autoclaving the media after filtering causes precipitates to form, making it difficult to tell if the media becomes contaminated.