Monthly Archives: May 2017

Preparation of Lambda broth

Overview

  • Lambda Broth is a moderately rich medium.
  • It is useful in the propagation of λ Phage.
  • It contains tryptone and sodium chloride (NaCl).
  • Tryptone provides basic nutrients and growth factors, thus support growth.
  • Sodium chloride (NaCl) maintains proper isotonic environment of the broth.

Requirements

  • Reagents

      • Tryptone
      • 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

Composition

  • 1% Tryptone
  • 0.25% Sodium chloride
  • pH : 7.0 ± 0.2

Objective

  • Preparation of 1000 ml Lambda broth

Preparation

Step 1: To prepare 1000 ml of Lambda broth, weigh out 10 grams tryptone and 2.5 grams sodium chloride in 2 litre conical flask/beaker. Add 800 ml deionized/Milli-Q water. Mix it using magnetic stirrer. 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 an option. 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 Lambda Broth 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 the table to prepare Lambda 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 gm 5 gm 10 gm 100 gm
Sodium chloride 0.25 gm 1.25 gm 2.5 gm 25 gm
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

 

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

Preparation of LB (Luria-Bertani), Lennox broth

Overview

  • 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.

Requirement

    • Reagents
      • Tryptone
      • 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

Composition

  • 1% Bacto-tryptone
  • 0.5% Bacto-yeast extract
  • 0.5% Sodium chloride
  • pH : 7.0 ± 0.2

Objective

  • Preparation of 1000 ml LB (Luria-Bertani), Miller broth

Preparation

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.

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.

Notes:

  • 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 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

 

Preparation of 2 X YT medium

Overview

  • 2 X YT medium is also known as 2 X TY medium
  • It is a rich medium.
  • It is recommended for the growth and propagation of E. coli infected with the single strand filamentous bacteriophage M13.
  • It contains tryptone, yeast extract, and sodium chloride.
  • Tryptone is a pancreatic digest of casein (also called Peptone C). It functions as an amino acid source for microorganisms.
  • Vitamins and certain trace elements are provided by yeast extract.
  • Sodium chloride (NaCl) maintains proper isotonic environment of the broth.

Requirement

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

Composition

  • 1.6% Tryptone
  • 1% Yeast extract
  • 0.5% Sodium chloride (NaCl)
  • pH : 7.0 ± 0.2

Objective

  • Preparation of 1000 ml 2 X YT medium

Preparation

Step 1: To prepare 1000 ml of 2 X YT medium, weigh out 16 grams tryptone, 10 grams yeast extract, 5 grams Sodium chloride (NaCl) in 2 litre conical flask/beaker. Add 800 ml deionized/Milli-Q water and mix it. Solution will appear translucent yellowish with undissolved media ingredients.

Tips:
  • One can use manual shaking using a glass pipette to mix the ingredients. 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 2 X YT 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.

Notes:

  • 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 2 X YT medium 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
Bacto-tryptone 1.6 gm 8 gm 16 gm 160 gm
Bacto-yeast extract 1 gm 5 gm 10 gm 100 gm
Sodium chloride 0.5 gm 2.5 gm 5 gm 50 gm
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