LB 1.5% Agar Plates without and with Antibiotic Supplements
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1. Place a magnetic stirring bar (50-mm) in a 2-L Erlenmeyer flask and add the following:
Reagent Quantity Agar (BD Bacto, dehydrated, BD 214010) 15 g Tryptone (BD Bacto, BD 211705) 10 g Yeast extract (BD Bacto, BD 212750) 5 g NaCl (Merck, 1.06404) 5 g H2O 1000 mL -
2. Mix briefly and autoclave for 20 min at 120°C and 15 psi.
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3. Place the flask on a magnetic stirrer and stir at low speed. Avoid stirring too fast as this generates bubbles that are difficult to remove in Step 6.
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4. Wait for the liquid to cool down until the flask can be barely touched with one's hand (a temperature of ∼60°C).
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5. If desired, while stirring, add 1 mL of a sterile 1000× stock solution of the appropriate antibiotic.
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6. Pour the melted medium into Petri dishes (100-mm × 15-mm; Fisher Scientific FB0875713). Aim to pour about 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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7. 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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8. Leave the plates at room temperature for 3 to 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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9. Store unsupplemented LB plates for up to 6 mo at 4°C.
