Preparation of TYGPN Medium

OVERVIEW

TYGPN is a rich microbial culture medium. It can be prepared by dissolving 20 g Tryptone, 10 g yeast extract, 10 g KNO3, 5 g Na2HPO4, 10 ml 80% glycerol  in water to a final volume of 1000 ml (Elbing & Brent, 2019).

REQUIREMENTS

Reagents and Solutions
Tryptone
Yeast extract
Potassium nitrate (KNO₃, Molecular Weight: 101.10)
Disodium hydrogen phosphate, anhydrous (Na₂HPO₄, Molecular Weight: 141.96)
80% Glycerol
Deionized / Milli-Q water

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

Composition
2% Tryptone
1% Yeast extract
98.91 mM KNO₃
35.22 mM Na₂HPO₄

Objective: 
Preparation of 1000 ml TYGPN medium

PREPARATION

Step 1: To prepare 1000 ml of TYGPN medium, weigh out 20 g Tryptone, 10 g yeast extract, 10 g KNO3, 5 g Na2HPO4 in a 2-liter Erlenmeyer flask. Add 800 ml deionized/Milli-Q water. Mix it using a magnetic stirrer.

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

Tip:
One can use manual shaking using a glass pipette to mix all 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 dissolved ingredients will appear as a transparent yellowish solution.

Precautions:
◊ Stop heating once all the ingredients are dissolved. Don’t unnecessarily heat the medium.
◊ Make sure to dissolve any residual powder sticking to the glass.

Step 3: Add 10 ml of 80% Glycerol and adjust the volume to 1000 ml with deionized / distilled water. Mix it again.

Step 4: Transfer the medium to autoclavable bottles or insert the cotton plug and cover the mouth of Erlenmeyer flask with aluminum foil.

Tip:
One can make small aliquots of the medium if needed (e.g., 5 ml aliquot in 50 ml Erlenmeyer flask).

Step 5: Sterilize the solution by autoclaving for 20 minutes at 15 lb/in2 (1.05 kg/cm2) on liquid cycle.

Note:
Elbing & Brent, 2019 recommend autoclaving for 25 minutes at 15 lb/in2 (1.05 kg/cm2).

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