- 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.
- 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
- 1.6% Tryptone
- 1% Yeast extract
- 0.5% Sodium chloride (NaCl)
- pH : 7.0 ± 0.2
- Preparation of 1000 ml 2 X YT medium
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.
- One can use manual shaking using a glass pipette to mix the ingredients. 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 2 X YT 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 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|