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Agar plates

Overview

Required volumes:

  • For a small Petri dish (90 mm): ~20 mL of agar mix
  • For a big plate (150 mm): ~50 mL of agar mix

Steps

  1. Add the following to a 500 mL or 1 L bottle (no need to mix) and autoclave:
Component Calculation Example amounts
LB mix (g) .025 x V 2.5
Bacteriological agar (g) .015 x V 1.5
Milli-Q (mL) V 100
  1. Prepare the following while waiting:
    1. Warm a water bath to 60C. This temperature should not break antibiotics and keep the mix liquid.
    2. Wipe off the plating area with 70% ethanol.
    3. Label each plate with antibiotics that were used, your initials, and data.
  2. Once autoclaving is done, stir the mixture, submerge in a water bath, and wait for 5 min for it to cool down (or to re-liquify).
    • You should be able to just hold the bottle in your hand for a few seconds.
  3. Light up a Bunsen burner.
    • You may also want to wear a face mask.
  4. (Optional) Add an appropriate antibiotic (1 uL of antibiotic per 1 mL of mix).
    • Swirl immediately after pouring the antibiotic so that the mix doesn't start gelatinizing locally.
  5. (Optional) For blue/white screening, add 70 ug/mL X-gal and 80 uM IPTG.
  6. Pour the mix to about .5 cm mark of a plate, immediately covering it.
    • Swirl the mix after pouring each plate to remove bubbles.
    • If the mix starts solidifying in the bottle, re-liquefy it on a low power in a microwave (only when not using antibiotics). Beware of boiling it.
  7. Wait ~30-60 min for the mix to solidify, then place under UV for 30 min with lids open.
    • It might be necessary to wipe off vapor from the lids. Use a paper towel.
  8. Test the plates by streaking a resistant strain on one and a non-resistant one on another and incubating overnight.
  9. Use in a few days (storing at room temperature) or place in a plastic bag and store at +4C.

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