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