Minimal E Medium Plates
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1. Pipette 20 mL of E medium salts stock (50×) into a 1-L Erlenmeyer flask (flask A). Add 480 mL of H2O and briefly mix by shaking the flask by hand.
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2. Place a magnetic stirring bar (50-mm) in a second 1-L Erlenmeyer flask (flask B). Add 15 g of Bacto Agar (BD 214010) and 500 mL of H2O.
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3. Autoclave flask A and flask B for 20 min at 120°C and 15 psi.
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4. Place flask B on a magnetic stirrer and stir at low speed.
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5. Wait for the liquid in flask A and flask B to cool down until the flasks can be barely touched with one's hand (a temperature of ∼60°C).
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6. Add 4 mL of sterile 50% (w/v) Glucose stock solution to flask A and briefly mix by shaking the flask by hand.
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7. Pour the contents of flask A into flask B while stirring. Avoid stirring too fast as this generates bubbles that are difficult to remove in Step 9.
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8. If desired, while stirring add the appropriate amount of a sterile solution of a supplement.
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9. Pour the melted medium into Petri dishes (100-mm × 15-mm; Fisher Scientific FB0875713). Aim to pour ∼25 mL per plate. With a little practice one will be able to gauge this amount. Should bubbles form on the surface of the plates, quickly apply the flame of a Bunsen burner (FIREBOY safety, Integra 144000) (or even that of a match) on the surface of the plate to pop the bubbles. Be careful not to melt the plastic of the dishes in the process.
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10. Once the agar has hardened, turn the plates upside down, stacking them in piles. Inverting the plates prevents vapor condensation on the lids and H2O droplets from falling on the surface of the agar.
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11. Leave the plates at room temperature for 3–4 d to allow them to dry slightly. For faster drying, place the plates in a laminar flow hood with the lids slightly ajar for 1–2 h.
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12. Store Minimal E medium plates for up to 6 mo at 4°C.
