Preparation of TNT Medium

OVERVIEW

• TNT medium is a moderately rich medium.
• It contains tryptone, sodium chloride (NaCl) and thiamine.
• Tryptone provides basic nutrients and growth factors, thus supporting growth.
• NaCl maintains a proper isotonic environment of the broth.
• It can be prepared by dissolving 10 g Tryptone, 5 g NaCl, and 0.1 mg Thiamine in water to a final volume of 1000 ml (Elbing & Brent, 2019).
  

REQUIREMENTS

Reagents and Solutions
Tryptone
Sodium chloride
1 mg/ml Thiamine solution
5N NaOH (for pH adjustment)
Deionized / Milli-Q water

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

Composition
1% Tryptone
0.5% Sodium chloride
0.1 µg/ml Thimine
pH : 7.3

Objective: 
Preparation of 1000 ml TNT medium

PREPARATION

Step 1: To prepare 1000 ml of TNT medium, weigh out 10 g tryptone and 5 g NaCl 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:
◊ Once all the ingredients of the medium dissolve 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 7.3 with 5N NaOH.

Note:
The TNT 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 down to 25°C (room temperature).

Step 4: Adjust the volume to 1000 ml with deionized / distilled water. Mix it again.

Step 5: Transfer the medium to autoclavable bottles 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 aliquot in 50 ml Erlenmeyer flask).

Step 6: Sterilize the solution by autoclaving for 20 minutes at 15 lb/in² (1.05 kg/cm²) on liquid cycle.

Note:
Elbing & Brent, 2019 suggest autoclaving for 25 min.

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.

Step 7: Cool down the solution to >50°C. Add 0.1 ml of 1mg/ml stock solution of Thiamine aseptically.

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, PMC