Preparation of 2 X YT Medium

Overview

The 2 x YT medium, also known as 2 x TY medium, 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 a proper isotonic environment of the broth.

It can be prepared by dissolving 16 g tryptone, 10 g yeast extract, and 5 g Sodium chloride (NaCl) in water to a final volume of 1000 ml (Elbing & Brent, 2019).

REQUIREMENT

Reagents and solutions
Tryptone
Yeast extract
Sodium chloride (NaCl)
5N NaOH (for pH adjustment)
Deionized / Distilled water

Equipment and disposables
Measuring cylinder
Erlenmeyer flask
Hot plate / Magnetic stirrer with a 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

PROCEDURE

Step 1: To prepare 1000 ml of 2 X YT medium, weigh out 16 g tryptone, 10 g yeast extract, 5 g Sodium chloride (NaCl) in a 2-liter Erlenmeyer flask. Add 800 ml deionized/Milli-Q water and mix it.

Note:
The solution will appear translucent-yellowish with undissolved medium ingredients.

Tip:
One can use manual shaking using a glass pipette to mix the ingredients. Mixing using a Magnetic stirrer is an option. The magnetic stirrer makes the dissolving process easy and convenient.

Precautions:
◊ Do not dissolve in 1000 ml of deionized / distilled water. It is always a good practice to dissolve all media ingredients and then adjust the volume to the desired volume with the solvent. In most cases, solution volume increases when a large amount of solute dissolves in the solvent.
◊ Make sure no clumps remain after making the suspension.

Step 2: Dissolve all ingredients completely by heating to boiling while stirring.

Note:
Medium with completely disolved ingredients will appear as a transparent yellowish solution.

Precautions:
◊ Once all the ingredients of the medium are dissolved and the medium appears transparent, stop the heating. Don’t unnecessarily 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).

Note:
The 2 X YT medium is not very highly buffered. The pH of the medium drops, as the growing culture reaches near the saturation phase.

Precaution:
Since pH is dependent on temperature, It is always a good practice 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 an autoclavable bottle or insert the cotton plug and cover the mouth Erlenmeyer flask with aluminum foil.

Tip:
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 for 20 minutes at 15 lb/in² (1.05 kg/cm²) 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 temperatures.
◊ After autoclaving, all sterile solutions should be handled inside the laminar flow hood to maintain sterility.

Storage
The solution can be stored at room temperature for a few days. Keep the medium at 2 – 8 °C for longer storage. 

Applications
Bacterial growth medium

REFERENCES

Elbing & Brent, 2019. Recipes and Tools for Culture of Escherichia coli. Curr Protoc Mol Biol. 125(1), e83. PMID-30412361; Full-Text Links: wiley, PMC6819147