Terrific Broth (TB) preparation

Terrific Broth (TB)

12 g/L tryptone (1.2% m/V)

24 g/L yeast extract (2.4% m/V)

4 mL/L glycerol (4.34 mM, 0.4% V/V)

17 mM KH2PO4, 2.31 g/L

72 mM K2HPO4, 12.54 g/L

(opt.) 15 g/L agar (1.5% m/V)

(opt., nontrad.) 5–10 mM 1 M MgSO4  or MgCl2

The original suggestion was to autoclave phosphate buffer and agar separately. However, it has recently been discovered that when combined, phosphate and agar react to produce H2O2. Fortunately, this reaction does not greatly affect E. coli due to their high catalase expression. Despite this, commercial mixes do not separate the two components and still recommend autoclaving. It is also worth noting that autoclaving glycerol separately is necessary because it can participate in Maillard reactions with amines found in tryptone/yeast extract. Although glycerol has fewer carbonyl groups, which contribute to Maillard reactions, it can still sequester nutrients and form growth-inhibitory compounds.

LB medium lacks sufficient magnesium. It has been observed that in peptone and tryptone broths, robust growth is dependent on the presence of Mg2+, especially when supplemented with glucose. Even in phosphate-buffered tryptone broth, the addition of 1 mM MgCl2 leads to significant growth improvement, which indicates the insufficient amount of contaminant Mg2+ in phosphate salts. However, phosphate does introduce substantial amounts of other trace minerals. To achieve optimal growth and minimize cell stress, it is recommended to supplement any peptide broth with 1–10 mM MgSO4 or MgCl2.

Souce: NovoPro    2023-07-18