Preparation of Luria-Bertani (LB), Miller Broth

OVERVIEW

• Luria-Bertani (LB) medium is a rich medium, very commonly used to grow E. coli.
• It contains tryptone, yeast extract, and sodium chloride (NaCl).
• Two LB (Luria-Bertani) medium, Miller broth and Lennox broth, are available which differ only in the concentration of sodium chloride.
• Tryptone provides basic nutrients and growth factors to the bacteria.
• Yeast extract supplies vitamins, amino acids, and trace elements.
• NaCl maintains the proper isotonic environment of the broth.
• Miller LB Broth contains twice the concentration of NaCl present in Lennox LB Broth.
• This allows the researcher to select the optimal salt concentration for the growth of a specific strain of E. coli.

REQUIREMENTS

Reagents and Solutions
> Tryptone
> Yeast extract
> Sodium chloride
> 5N NaOH (for pH adjustment)
> Deionized / Milli-Q water

Equipment and disposables
Measuring cylinder
Conical flask / Beaker
Hotplate / Magnetic stirrer with heating device

Equipment and disposables
Measuring cylinder
Conical flask / Beaker
Hotplate / Magnetic stirrer with heating device

Composition
1% Bacto-tryptone
0.5% Bacto-yeast extract
1% Sodium chloride (NaCl)
pH : 7.0 ± 0.2

Objective
Preparation of 1000 ml LB (Luria-Bertani), Miller broth
Note: LB (Luria-Bertani), Miller broth is commonly known as LB (Luria-Bertani) medium.

PREPARATION

Step 1: To prepare 1000 ml of Miller LB broth, weigh out 10 g tryptone, 5 g yeast extract, and 10 g NaCl in a 2-liter conical flask/beaker. Add 800 ml deionized/Milli-Q water. Mix it. The solution will appear translucent yellowish with undissolved media ingredients.

Tip:
One can use manual shaking using a glass pipette to mix all ingredients. Mixing using a Magnetic stirrer is optional. The magnetic stirrer makes the dissolving process easy and convenient.

Precautions:
1. Do not dissolve in 1000 ml of deionized / Milli-Q water. It is always a good practice to dissolve all media ingredients first and then adjust the volume to the desired volume with the solvent. In most cases, solution volume increases when a large amount of solute dissolves in the solvent.
2. Make sure no lumps are remaining after making the suspension of medium components.

Step 2: Dissolve all ingredients completely by heating to boiling while stirring.

Note:
Medium with completely dissolved ingredients will appear as a transparent yellowish solution.

Precautions:
1. Once all the ingredients of the medium are dissolved and the medium appears transparent, stop the heating. Don’t unnecessarily heat the medium.
2. Make sure to dissolve any residual powder sticking to the glass.

Step 3: Cool down the medium to room temperature. Adjust the pH 7.0 with 5N NaOH (~0.2 ml).

Note:
The LB medium is not very highly buffered. The pH of the medium drops, as the growing culture reaches near the saturation phase.

Precaution:
Since pH is dependent on temperature, It is always a good practice to adjust the medium pH only after the solution has cooled down to 25°C (room temperature).

Step 4: Adjust the volume to 1000 ml with deionized / Milli-Q water. Mix it again.

Step 5: Transfer the medium to autoclavable bottles or cover the Erlenmeyer flask with cotton plug and aluminum foil.

Tip:
One can make small aliquots of the medium if neeed (e.g., 5 ml aliquot in 50 ml conical flask).

Step 6: Sterilize the solution by autoclaving for 20 minutes at 15 lb/in² (1.05 kg/cm²) on liquid cycle.

Precautions:
1. Antibiotics and nutritional supplements should be added only after the solution has cooled to 40°C or below. Many supplements, like antibiotics, are degraded at high temperatures.
2. After autoclaving, all sterile solutions should be handled inside the laminar flow hood to maintain sterility.

Storage
The solution can be stored at room temperature for a few days. Keep the medium at 2 – 8 °C for longer storage. 

Applications
Bacterial growth medium