Choano Media

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Contents 1 Cereal Grass Choano Medium 2 WC-Media 3 Artificial Seawater for Loricate Choanoflagellates 4 Pratt's medium for freshwater flagellates 5 Schmaltz-Pratt medium for marine flagellates

Cereal Grass Choano Medium

Acronym: CGM (cereal grass medium)

Species: Monosiga brevicollis, Proterospongia sp. ATCC 50818

Written by: Stephen Fairclough, Mark Dayel

This recipe is based on ATCC medium 1525.

Media Materials

• 4L Erlenmeyer flask
• 4L vaccum flask (side-arm flask)
• Natural seawater or artificial sea water (Tropic Marin Sea Salts) prepared with Milli-Q water. (I also tested Instant Ocean and seem to work as well. Mx2 Growth Curve)
• Cereal grass (see Cereal grass sources)
• Buchner (ceramic) funnel
• #1 Whatman paper (cat# 1001-150)
• 0.22-micron sterile filters (perferably Millipore Steritop/cup)

Preparation Protocol

1. Boil 2L natural seawater by autoclaving for 40min
2. Add 10g of cereal grass to boiled sea water
3. Allow to steep until cool (~3-5hrs or overnight)
4. Remove macroscopic cereal grass particles by vacuum filtering twice through Buchner funnel with a double layer of #1 Whatman paper
5. Sterile filter through a .22-micron filter in a laminar flow hood

Notes on making cereal grass medium

After testing a variety of filtering devices we have found that Millipore Steritops/cups 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. I find the using Millipore Stericups is better than using Steritops in to 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 causes precipitates to form making it difficult to tell if the media becomes contaminated.

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WC-Media

Freshwater media for choanos

Acronym: WC

Species:

Written by: Frank Nitsche

Modified from R.R.L. Guillard and C.J. Lorenzen 1972.

Preparation Protocol

1. Prepare each of the stock solutions (see below) and add 1ml of each to 1 litre of destilled water + TES buffer
2. Autoclave and add 1ml of the micronutrient solution and 1ml of the vitamin solution
3. Add either quinoa seeds or a wheat grain as carbon source for bacteria per culture flask

stock solutions

[g/1000 ml dist. H2O]

• CaCl₂ . 2H₂O (36.8g)
• MgSO₄ . 7H₂O (37g)
• NaHCO₃ (12.6g)
• K₂HPO₄ . 3H₂O (11.4g)
• NaNO₃ (85g)
• Na₂SiO₃ . 9H₂O (21.2 g)
• TES buffer (0.115g)

micronutrient solution

[g/1000 ml dist. H2O]

• Na₂EDTA (4.36g)
• FeCl₃ . 6H₂O (3.15g)
• CuSO₄ . 5H₂O (0.01g)
• ZnSO₄ .7H₂O (0.022g)
• CoCl₂ . 6H₂O (0.01g)
• MnCl₂ . 4H₂O (0.18g)
• Na₂MoO₄ . 2H₂O (0.006g)
• H₃BO₃ (1g)

vitamin solution !!!store at 4°C!!!

[g/1000 ml dist. H2O]

• thiamin HCl (0.1g)
• biotin (0.0005g)

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Artificial Seawater for Loricate Choanoflagellates

Acronym: ASW (artificial sea water)

Species: Stephanoeca diplocostata, Diaphanoeca grandis

Written by: Alan Marron 8th July 2009

Media Materials

• 1L Schott Bottle
• Tropic Marin Sea Salts
• dd Water
• Hydrometer
• Steriop presterlized 0.22-micron filter
• Vacuum Pump
• Rice
• Autoclave bags
• Autoclave tape

Preparation Protocol

1. Add 35.6g of Tropic Marin Sea Salts to 1L of dd Water
2. Measure the specific gravity of the artificial seawater using a hydrometer (I use a Coralife Deep 6 Hydrometer). Adjust the salt concentration until the specific gravity is between 1.020-1.023
3. Connect the 1L Schott Bottle to the screw-in base of the Steriop 0.22 micron filter
4. Connect the side arm of the Steriop 0.22micron filter to the vacuum pump
5. Pour the artificial seawater into the filter reservoir and turn on the pump until the Schott bottle is filled
6. Disconnect the Schott bottle and pump. The Steriop filter may be used again for up to 6-7L of artificial seawater until the salt clogs the pores and reduces filter quality
7. Place rice grains into the autoclave bag
8. Fold over the open end of the autoclave bag such that the rice is sealed with minimum air in the pocket. This ensures that there is a minimum of water vapour from the air so that the rice is autoclaved dry
9. Seal with autoclave tape
10. Autoclave the full Schott bottle and autoclave bag
11. When finished, allow both to cool at the temperature at which the choanos are being cultured. This avoids any temperature stress to the choanos when making new cultures
12. Only open the rice or seawater under a laminar flow hood to stop any bacterial contamination

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Pratt's medium for freshwater flagellates

Acronym:

Species:

Contributed by: S. Karpov

Recipe

• KNO₃ - 0.1 g/L of distilled water
• MgSO₄ . 7 H₂O - 0.01 g/L
• K₂HPO₄ . 3 H₂O - 0.01 g/L
• FeCl₃ . 6 H₂O - 0.001 g/L

pH = 6.5-7.5

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Schmaltz-Pratt medium for marine flagellates

Acronym:

Species:

Contributed by: S. Karpov

Recipe for salinity 20%

• NaCl 16.07 g/L of distilled water
• KCl 0.38 g/L
• MgCl₂ . 6 H₂O 3.15 g/L
• MgSO₄ . 7 H₂O 3.95 g/L
• CaCl₂ . H₂O 0.83 g/L
• KNO₃ 0.06 g/L
• K₂HPO₄ . 3 H₂O 0.006 g/L

Bacterial food source: Pseudomonas fluorescens

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