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Materials and Methodology
Materials
- 12 11 x 6 cm (4"-5") clay pots /site
- 12 plastic petri dishes/ site (6 for agar pot construction & 6 for lab work)
- Silicone adhesive without fungicide with caulking gun
- Rubber stoppers for each clay pot (pencil end erasers work)
- Agar medium – 1.5% or 15 gr/L distilled H2O
- Distilled water
- NaNO3 - 0.5 M
- KH2PO4 - 0.5 M
- Nylon cord and attachment pegs (anchors)
- Surveyor tape or ribbon/stakes
- Zip lock bags – 1 quart (one per pot)
Equipment
- 4 2- liter Erlenmeyer flasks
- 3 Scoops
- Magnetic stirring hotplates
- Thermometer
- Funnel(s)
- Hot pads or mitts
- 3 Balances
- Several smooth bladed knives or small scrapers
- Wading boots
- Latex gloves
- Small plastic tubs
Lab preparation of agar pots and nutrient agar mixtures
1. Soak the clay pots in distilled water for 2 weeks. Air-dry and run a bead of silicone caulk around the edge of each pot. Adhere the lipped edge of the petri dish to the
bottom of the clay pot creating a nutrient diffusing substrata. (Scrimgeour and Chambers, 1997)
2. Mix the nutrient medium in 1-liter batches.
Nutrient agar medium recipes for 36 pots [enough for 3 sites/ 9 groups]
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Batch
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1.5% agar
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NaNO3
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KH2P04
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Yield
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Control
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2L
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2L
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Nitrate
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2L
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42.5 g/L
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2L
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Phosphate
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2L
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68.9 g/L
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2L
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N + P
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2L
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42.5 g/L
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68.9 g/L
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2L
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Combine designated ingredients in an Erlenmeyer flask and heat on a stirring hotplate until the agar comes to a boil. Remove immediately. Allow the agar to
cool to 700 C., then pour into each pot. Gently tap each pot after filling to release any tapped air bubbles. Each pot holds approximately 170 ml of agar medium. As necessary, top off the pots with agar.
Alternate mixing protocol: Mix agar and water in 1-liter flasks on stirring hotplates. Heat solution to 90 degrees C. for 20 minutes to ensure that agar is fully
dissolved. Cool to 700 C. Add other nutrients in designated batches gradually over 5 minutes, stirring for an additional 10 minutes to allow nutrients to dissolve fully. (Scrimgeour and Chambers, 1997).
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