Preparation of Superbroth

Overview

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).

REQUIREMENTS

Reagents and Solutions
Tryptone
Yeast extract

Sodium chloride
1N NaOH
Deionized / Milli-Q water

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

Composition
3.2 % Tryptone

2% Yeast extract
0.5% Sodium chloride
pH : 7.0 ± 0.2

Objective: 
Preparation of 1000 ml Superbroth

PREPARATION

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.

Note:
The solution will appear translucent-dark brown 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 lumps 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 dark brown 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: 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.

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 recommend autoclaving medium for 25 min.
Cshprotocols recommend autoclaving medium for 20 min at 15 psi.

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

Follow the table to prepare Superbroth of various volumes.
Reagents / Volume100 ml500 ml1,000 ml10,000 ml
Tryptone
3.2 g16 g
32 g
320 g
Yeast extract
2 g
10 g
20 g
200 g
Sodium chloride0.5 g
2.5 g
5 g
50 g
1 N NaOH0.5 ml
2.5 ml
5 ml
50 ml
WaterAdjust the final volume to 100 mlAdjust the final volume to 500 mlAdjust the final volume to 1000 mlAdjust the final volume to 10000 ml

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
  • http://cshprotocols.cshlp.org/content/2006/1/pdb.rec453

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