Sand agar plates
MATERIALS
Reagents
Bacto agar (15 g)
Bold’s modified basal freshwater nutrient (BMBFN) medium (Sigma-Aldrich) (20 mL)
Ottawa sand (granular silicon dioxide [SiO2], sterilized) (Sigma-Aldrich)
Equipment
Autoclave
Bunsen burner
Flask, graduated
Hood (laminar flow)
Petri dishes (90 mm)
Stirrer
METHOD
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1. Mix 15 g Bacto agar in 965 mL of H2O in a flask.
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2. Add 20 mL of BMBFN medium, shake and heat until the agar has dissolved. Adjust the pH to 7.0 with 5 N NaOH (~0.2 mL) or HCl. Adjust the final volume of the solution to 1 L with H2O.
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3. Sterilize by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle. When the medium is removed from the autoclave, swirl it gently to distribute the melted agar evenly throughout the solution. Be careful! The fluid may be superheated and may boil over when swirled. Before pouring the plates, allow the medium to cool to 50°C.
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4. Pour plates directly from the flask; allow ~30-35 mL of medium per 90-mm Petri dish. Line up five poured plates and spread ~0.2 g of sterilized Ottawa sand evenly over the plates. To remove bubbles from the medium in the plate, flame the surface of the medium with a Bunsen burner before the agar hardens.
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5. Leave the Petri dishes in the laminar flow hood until the medium is solid and there is no condensation on the lids. When the medium has hardened completely, invert the plates and store them at 4°C until needed.
