- LB (Luria-Bertani) medium, a rich medium,is 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.
- Lennox LB Broth contains half the concentration of sodium chloride present in Miller LB Broth.
- This allows the researcher to select the optimal salt concentration for the growth of a specific strain of E. coli.
- Yeast extract
- Sodium chloride
- 5N NaOH (for pH adjustment)
- Deionized / Milli-Q water
- Equipment and disposable
- Measuring cylinder
- Conical flask / Beaker
- Hot plate / Magnetic stirrer with heating device
- 1% Bacto-tryptone
- 0.5% Bacto-yeast extract
- 0.5% Sodium chloride
- pH : 7.0 ± 0.2
- Preparation of 1000 ml LB (Luria-Bertani), Miller broth
Step 1: To prepare 1000 ml of Lennox 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.
- 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.
- 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.
- Medium with completely dissolved ingredients will appear as transparent yellowish solution.
- 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).
- The LB medium is not very highly buffered. The pH of medium drops, as the growing culture reaches near saturation phase.
- 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.
- 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).
- 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.
- Solution can be stored at room temperature for few days. Keep the medium at 2 – 8 °C for longer storage.
- Bacterial growth medium
|Follow table to prepare Lennox 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||0.5 g||2.5 g||5 g||50 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