Terrific Broth

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    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.

    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.

    Composition

    IngredientsMolarityFor 100mLFor 500mLFor 1000ml
    Bacto Tryptone 1.2 gm6 gm12 gm
    Yeast extract2.4 gm12 gm24 gm
    Potassium Phosphate, Dibasic (K2HPO4)72mM (174.18 MW)1.25 gm6.25 gm12.5 gm
    Potassium Phosphate, Monobasic (KH2PO4)17mM (136.09 MW)0.23 gm1.15 gm2.3 gm
    Glycerol0.4 ml2 ml4 ml
    Agar1.5 gm7.5 gm15 gm
    Distilled waterUp to 100 mlUp to 500 mlUp to 1000 ml

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

    IngredientsBacto TryptoneYeast extractPotassium Phosphate, Dibasic (K2HPO4)Potassium Phosphate, Monobasic (KH2PO4)Glycerol
    CSHL20 gm24 gm12.5 gm2.3 gm4 ml
    MERCK11.8 gm23.6 gm9.4 gm2.2 gm4 ml
    helmholtz-muenchen.de12 gm24 gm12.5 gm2.3 gm5 ml

    Reference

    1. Merck Microbiology Manual 12th Edition
    2. Thermofisher
    3. MPBIO

     

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