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).
Reagents and Solutions
Potassium nitrate (KNO3, Molecular Weight: 101.10)
Disodium hydrogen phosphate, anhydrous (Na2HPO4, Molecular Weight: 141.96)
Deionized / Milli-Q water
Equipment and disposables
Hot plate / Magnetic stirrer with heating device
1% Yeast extract
98.91 mM KNO3
35.22 mM Na2HPO4
Preparation of 1000 ml TYGPN medium
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.
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.
◊ 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.
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.
Elbing & Brent, 2019 recommend autoclaving for 25 minutes at 15 lb/in2 (1.05 kg/cm2).
◊ 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.
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 TYGPN Medium of various volumes.|
|Reagents / Volume||100 ml||500 ml||1,000 ml||10,000 ml|
|Tryptone||2 g||10 g||20 g||200 g|
|Yeast extract||1 g||5 g||10 g||100 g|
|KNO3||1 g||5 g||10 g||100 g|
|Na2HPO4||0.5 g||2.5 g||5 g||50 g|
|80% Glycerol||1 ml||5 ml||10 ml||100 ml|
|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|