Terrific Broth

Introduction

Terrific broth, developed by Tartoff and Hobbs, is a buffered and enriched medium used to cultivate recombinant strains of E.coli. It is a nutrient-rich medium containing glycerol, peptides, amino acids, water-soluble vitamins, and carbohydrates. Terrific broth (TB) contains more tryptone and yeast extract than the standard LB medium. Terrific broth can be used for almost all applications involving liquid bacterial culturing steps such as protein expression and cloning.

Principle

The terrific broth is a nutrient-rich growth medium that supports the high cell density of E.coli. It maintains growth in the logarithmic phase resulting in larger yields of plasmid DNA and heterologous proteins. Tryptone and yeast extract act as the sources of carbon, nitrogen, vitamins, and amino acids required for the growth of bacteria. The potassium phosphate buffer system acts as the source of potassium and prevents cell death and, eventually, the drop in pH. Glycerol is added to the medium instead of glucose as a carbon source. Glycerol is not reduced to acetic acid. Glucose acts as a repressor in various protein expression systems, whereas glycerol promotes growth.

Applications

It gives a higher plasmid yield per volume compared with LB media.
TB (not to be confused with T broth) can obtain higher yields of recombinant protein expression in E.coli. However, some proteins require slow E.coli growth for proper folding. For such cases, it would be better to use M9 media.

Composition

Ingredients	Molarity	For 100mL	For 500mL	For 1000ml
Bacto Tryptone		1.2 gm	6 gm	12 gm
Yeast extract		2.4 gm	12 gm	24 gm
Potassium Phosphate, Dibasic (K₂HPO₄)	72mM (174.18 MW)	1.25 gm	6.25 gm	12.5 gm
Potassium Phosphate, Monobasic (KH₂PO₄)	17mM (136.09 MW)	0.23 gm	1.15 gm	2.3 gm
Glycerol		0.4 ml	2 ml	4 ml
Agar		1.5 gm	7.5 gm	15 gm
Distilled water		Up to 100 ml	Up to 500 ml	Up to 1000 ml

Table 3. Composition of Terrific broth.

pH

The pH of the medium is adjusted to 7.2 ± 0.2 at 25°C. Adjust the pH with 1N HCl and 1N NaOH.

Preparation

About 50.8gm of dehydrated media is added to 900m of distilled water.
Add 4 ml of glycerol and mix well, and the final volume is adjusted to 1000 ml.
Boil for 1 minute and dissolve the contents properly with regular agitation. If all ingredients are adequately dissolved, you can skip this step.
Adjust the pH to 7.2.
Dispense the desired volume in proper containers.
Add agar powder (1-1.5%) if planned to make solid media.
Sterilize them by autoclaving at 121℃ for 15 to 20 minutes.
Add appropriate antibiotics to the media before solidifying to allow the selection of transformants.

Alternative preparation method: Apart from the workflow mentioned above, some labs use potassium salts prepared at 10x concentrate stored separately to add after autoclaving of other media components. The salt solution (buffer) needs to be sterilized by filtering or autoclave. This two-step process can control salt concentration if the experiment needs different salt concentrations.

Preparation of agar media

Add agar powder (1-1.5%) if planned to make solid media.

Storage

It is hygroscopic in nature and moisture-sensitive. After preparation, the medium is yellowish-brown in color and is stored at 2-8℃ in the refrigerator. It can be stored for about four weeks.

Variations in compositions

Like other cultural media, the terrific broth does come in different flavors. Following are a few different compositions with sources from which they were acquired.

Ingredients	Bacto Tryptone	Yeast extract	Potassium Phosphate, Dibasic (K₂HPO₄)	Potassium Phosphate, Monobasic (KH₂PO₄)	Glycerol
CSHL	20 gm	24 gm	12.5 gm	2.3 gm	4 ml
MERCK	11.8 gm	23.6 gm	9.4 gm	2.2 gm	4 ml
helmholtz-muenchen.de	12 gm	24 gm	12.5 gm	2.3 gm	5 ml

Table 2. Table showing composition differences from various sources.

Reference

Merck Microbiology Manual 12th Edition
Thermofisher
MPBIO

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