Preparation of LB-Agar Medium and Plates

OVERVIEW

LB medium can be solidified by adding agar. Solidified culture medium is very useful for the selection and identification of specific colonies of the bacteria harbouring desired traits.

Agar melts at 85°C but melton agar stays liquid at a temperature > 40°C (this property is called hysteresis, different melting and solidifying temperatures). This allows us to add heat-labile medium supplements such as antibiotics and vitamins to melton medium and prepare solid medium.

LB-agar solid medium is prepared by dissolving 15 g agar / liter of LB (Elbing & Brent, 2019). Whenever needed, solid medium is melted and plates can be poured.

Here we describe a procedure to prepare 500 ml Lambda-agar solid medium that is sufficient to prepare ≈ 15 plates (≈ 30 ml medium/10 cm petri-plate).

REQUIREMENTS

Reagents and Solutions
Lambda broth
Agar

Equipment and disposables
Erlenmeyer flask
Hot plate
10 cm Bacteriological Petri Dishes with Lid

Composition
1x Lambda broth
1.5% Agar

Objective: 
Preparation of 500 ml of LB-agar medium

PREPARATION

Step 1: Weigh out 7.5 g agar in a 1-liter Erlenmeyer flask/autoclavable bottle. Add 500 ml LB medium. Mix it by swirling the flask.

Step 2: Close the lid of the bottle loosely/insert a cotton plug and cover the mouth of the Erlenmeyer flask with aluminum foil.

Step 3: Sterilize the solution by autoclaving for 20 minutes at 15 lb/in2 (1.05 kg/cm2) on liquid cycle.

Important notes:
LB-agar medium (solid at room temperature) can be stored at room temperature for a few weeks but for a longer time, we recommend you to store it in the cold room. 

Depending on your convenience, you can prepare the LB-agar plate any time by melting the medium.

After autoclaving, the medium must be handled aseptically to maintain sterility.

Step 4: Allow the medium to cool below 50°C. If required, add supplements such as antibiotics. Mix by swirling the flask gently.

Step 5: Pour ≈ 30 ml medium per 10 cm plate. Let the medium solidify. Keep the plate open for 2 h at room temperature to dry the plate.

Tip:
Remove the trapped air by touching with the needle/micropipette tip.

Precaution:
Don’t cover the plate with a lid. If the plate is covered with a lid, all the vapour will condense on the lid and form water droplets that eventually fall back to the solidified medium.

Storage:
Cover the plates with their lid and store at 4°C in an inverted position.

REFERENCES

  • Elbing & Brent, 2019. Recipes and Tools for Culture of Escherichia coli. Curr Protoc Mol Biol. 125(1), e83. PMID-30412361; Full-Text Links: wiley, PMC

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