- 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).
Reagents and Solutions
1 mg/ml Thiamine solution
5N NaOH (for pH adjustment)
Deionized / Milli-Q water
Equipment and disposables
Hot plate / Magnetic stirrer with heating device
0.5% Sodium chloride
0.1 µg/ml Thimine
pH : 7.3
Preparation of 1000 ml TNT medium
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.
The solution will appear translucent-yellowish with undissolved media ingredients.
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.
◊ 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.
Medium with completely dissolved ingredients will appear as a transparent yellowish solution.
◊ 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.
The TNT medium is not very highly buffered. The pH of the medium drops, as the growing culture reaches near the saturation phase.
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.
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.
Elbing & Brent, 2019 suggest autoclaving for 25 min.
◊ 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.
The solution can be stored at room temperature for a few days. Keep the medium at 2 – 8 °C for longer storage.
Bacterial growth medium
|Follow the table to prepare Lambda broth of various volumes.|
|Reagents / Volume||100 ml||500 ml||1,000 ml||10,000 ml|
|Tryptone||1 g||5 g||10 g||100 g|
|Sodium chloride||0.5 g||2.5 g||2.5 g||25 g|
|1mg/ml Thimine||10 µL||50 µL||100 µL||1000 µL|
|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|
- 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