Preparation of LB (Luria-Bertani), Miller broth

Overview

  • LB (Luria-Bertani) 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.
  • Sodium chloride (NaCl) maintains the proper isotonic environment of the broth.
  • Miller LB Broth contains twice the concentration of sodium chloride 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.

Requirement

  • Reagents
    • Tryptone
    • Yeast extract
    • Sodium chloride
    • 5N NaOH (for pH adjustment)
    • Deionized / Milli-Q water
  • Equipment and disposable
    • Measuring cylinder
    • Conical flask / Beaker
    • Hotplate / Magnetic stirrer with heating device

Composition

  • 1% Bacto-tryptone
  • 0.5% Bacto-yeast extract
  • 1% Sodium chloride
  • 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 grams tryptone, 5 grams yeast extract and 10 grams sodium chloride in 2 litre conical flask / beaker. Add 800 ml deionized/Milli-Q water. Mix it. Solution will appear translucent yellowish with undissolved media ingredients.

Tips:

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

Precautions:

  • Do not dissolve in 1000 ml of deionized / Milli-Q water. In most cases, solution volume increases when the large amount of solute is dissolved in solvent.
  • 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.

Notes:

  • Medium with completely dissolved ingredients will appear as transparent yellowish solution.

Precautions:

  • Once all the ingredients of the medium are dissolved and medium appears transparent, stop the heating. Don’t unnecessary heat the medium.
  • 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).

Notes:

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

Precautions:

  • Since pH is dependent on temperature, It is important to adjust the medium pH only after the solution has cooled 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 bottle or cover the conical flask with cotton plug and aluminium foil.

Tips:

  • One can make small aliquots of the medium if needed (e.g., 5 ml aliquots in 50 ml conical flask).

Step 6: Sterilize the solution by autoclaving (20 minutes at 15 lb/sq.in. (psi) from 121-124°C on liquid cycle).

Precautions:

  • 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 temperature.
  • After autoclaving, all sterile solutions should be handled inside the laminar flow hood to maintain the sterility.

Storage

  • Solution can be stored at room temperature for few days. Keep the medium at 2 – 8 °C for longer storage.  

Applications

  • Bacterial growth medium
Follow table to prepare Miller LB broth of various volume (100 ml, 500 ml, 1,000 ml and 10,000 ml)..
Reagents / Volume 100 ml 500 ml 1,000 ml 10,000 ml
Tryptone 1 g 5 g 10 g 100 g
Yeast extract 0.5 g 2.5 g 5 g 50 g
Sodium chloride 1 g 5 g 10 g 100 g
Water Adjust the final volume to 100 ml Adjust the final volume to 500 ml Adjust the final volume to 1000 ml Adjust the final volume to 10000 ml