LB agar plates
Updated 7/20/2021 11:17 PM
Introduction
Materials
- Agar
- Tryptone
- NaCl
- Yeast Extract
- Distilled water
Procedure
- Weigh out 7.5g agar, 5g tryptone, 5g sodium chloride (NaCl) and 2.5g yeast extract and add to a 1L bottle.
- Measure out approximately 400mL of distilled water and add to the bottle.
- Shake the bottle to dissolve the reagents.
- Once the reagents have fully dissolved, adjust the pH to 7.0 by using sodium hydroxide (NaOH) solution.
- Once the pH is adjusted, top up the solution to 500mL by using distilled water. Optional: set a water bath to 55ºC.
- To sterilise, autoclave the solution on a liquid cycle (20 min at 15 psi).
- Leave the solution to cool to approximately 55ºC, or warm enough to be held in your hand without burning it. If you do not have enough time to keep an eye on the temperature, put the bottle into the water bath. This will prevent the solution from setting prematurely.
- When cooled, add the appropriate antibiotic into the solution and swirl to mix.
- Prepare Petri dishes in a sterile environment (surfaces cleaned with 70% ethanol etc.). Carefully pour a thin layer of solution into the Petri dishes to cover the bottom of the plate (approximately 10 – 20mL per plate). Try to avoid transferring or creating any bubbles.
- Leave the plates to set before storing them in the fridge. Stack the plates upside down in towers of 10 plates. Ideally, wrap the towers in the plastic sheeting that came with the Petri dishes. Store the plates in the fridge (+4ºC).
The recipe is used to prepare a 500mL LB agar solution. This amount will be enough to make approximately 20 – 30 plates.