Preparation of Terrific Broth (TB)

Overview:

The Terrific Broth was formulated by Tartoff and Hobbs (Tartoff & Hobbs, 1987). It is a rich medium, which is suitable for the rapid growth of E. coli. It contains tryptone, yeast extract, glycerol, potassium nitrate, and sodium phosphate.  Tryptone is a pancreatic digest of casein (also called Peptone C). It functions as an amino acid source for microorganisms. Vitamins and certain trace elements are provided by yeast extract. Glycerol is an additional source of carbon in Terrific Broth. Unlike glucose, it is not fermented to acetic acid.

The TB salt – Potassium phosphate dibasic and potassium phosphate monobasic functions as a buffer system that maintains the pH of the medium during the culture growth. This prevents the cell death of bacteria due to a drop in pH.

REQUIREMENTS

Reagents and solutions
Bacto-Tryptone
Bacto-Yeast extract
Glycerol (99 – 100%)
TB salt solution [170 mM Potassium phosphate, monobasic (KH2PO4), 720 mM Potassium phosphate, dibasic (K2HPO4)]
5N NaOH (for pH adjustment)
Deionized / Milli-Q water

Equipment and disposables
Measuring cylinder
Conical flask / Beaker
Hot plate / Magnetic stirrer with a heating device

Objective
Preparation of 1000 ml Terrific Broth

PROCEDURE

Step 1: To prepare 1000 ml of Terrific Broth, weigh out 12 g tryptone and 24 grams yeast extract in a 2-liter conical flask/beaker. Add 800 ml deionized/Milli-Q water and mix it. The solution will appear translucent yellowish with undissolved media ingredients.

Tip:
◊ One can use manual shaking using a glass pipette to mix the ingredients. Magnetic stirrer makes the dissolving process easy and convenient.

Precautions:
◊ Do not dissolve in 1000 ml of deionized / Milli-Q water. In most cases, solution volume increases when a large amount of solute is dissolved in a solvent.
◊ Make sure no lumps are remaining after making the suspension of medium components.

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 ingredients are dissolved 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. Add 4 ml of glycerol (99 – 100%) and mix. Adjust the pH 7.0 with 5N NaOH (~0.2 ml).

Step 4: Adjust the volume to 900 ml with deionized / Milli-Q water. Mix it again.

Step 5: Transfer the medium to an autoclavable bottle or cover the conical flask with a cotton plug and aluminum foil.

Step 6: Sterilize the solution by autoclaving (20 minutes at 15 lb/sq.in. (psi) from 121-124°C on liquid cycle). Allow the solution to cool to room temperature. Add 100 ml sterile TB salt solution. Mix it.

Precaution:
After autoclaving, all sterile solutions should be handled inside the laminar flow hood to maintain sterility.

Storage
Terrific Broth can be stored at room temperature for a few weeks. Keep the medium at 4 °C for longer storage.

Applications
Bacterial growth medium