Superbroth contains very high-levels of Tryptone and yeast extract that makes it a super rich medium. It can be prepared by dissolving 32 g tryptone, 20 g yeast extract, 5 g NaCl and 5 ml of 1 N NaOH in water to a final volume of 1000 ml (Elbing & Brent, 2019).
Reagents and Solutions
Deionized / Milli-Q water
Equipment and disposables
Hot plate / Magnetic stirrer with heating device
3.2 % Tryptone
2% Yeast extract
0.5% Sodium chloride
pH : 7.0 ± 0.2
Preparation of 1000 ml Superbroth
Step 1: To prepare 1000 ml of Superbroth, weigh out 32 g tryptone, 20 g yeast extract 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-dark brown 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 lumps 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 dark brown 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: Add 5 ml of 1N NaOH and adjust the volume to 1000 ml with deionized / distilled water. Mix it again.
Step 4: Transfer the medium to autoclavable bottles or cover the Erlenmeyer flask with cotton plug and 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.
◊ 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 Superbroth of various volumes.|
|Reagents / Volume||100 ml||500 ml||1,000 ml||10,000 ml|
|Tryptone ||3.2 g||16 g||32 g||320 g|
|Yeast extract||2 g||10 g||20 g||200 g|
|Sodium chloride||0.5 g||2.5 g||5 g||50 g|
|1 N NaOH||0.5 ml||2.5 ml||5 ml||50 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|
- 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