Lysogeny broth

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LB medium bottle and LB agar plate
LB medium bottle and LB agar plate

Lysogeny broth (LB), a nutritionally rich medium, is primarily used for the growth of bacteria. It is also known as Luria broth or Luria-Bertani broth.

LB media formulations have been an industry standard for the cultivation of Escherichia coli as far back as the 1950s. These media have been widely used in molecular microbiology applications for the preparation of plasmid DNA and recombinant proteins. It continues to be one of the most common media used for maintaining and cultivating recombinant strains of Escherichia coli.

There are several common formulations of LB. Although they are different, they generally share a somewhat similar composition of ingredients used to promote growth, including the following:

Peptides and peptones are provided by tryptone. Vitamins and certain trace elements are provided by yeast extract. Sodium ions for transport and osmotic balance are provided by sodium chloride. Bacto-tryptone is used to provide essential amino acids to the growing bacteria, while the bacto-yeast extract is used to provide a plethora of organic compounds helpful for bacterial growth.

Contents

[edit] Formulas

The formulations generally differ in the amount of sodium chloride, thus providing selection of the appropriate osmotic conditions for the particular bacterial strain and desired culture conditions. The low salt formulations, Lennox and Luria, are ideal for cultures requiring salt-sensitive antibiotics.

  • LB-Miller (10 g/l NaCl)
  • LB-Lennox (5 g/l NaCl)
  • LB-Luria (0.5 g/l NaCl)

[edit] Misnomers

LB is also known as:

  • Luria-Bertani broth (though this name is very widely used)
  • Luria broth
  • Lennox broth

The recipe for LB was formulated by Giuseppe Bertani and published in 1951. Over the years the acronym has been widely misconstrued. In the Postscript to his 2004 paper, "Lysogeny at Mid-Twentieth Century: P1, P2, and Other Experimental Systems", Giuseppe Bertani clarified the original meaning of the acronym:

"My first paper on lysogeny, describing the modified single-burst experiment and the isolation of P1, P2, and P3, also contained the formula of the LB medium which I had concocted in order to optimize Shigella growth and plaque formation. Its use has since become very popular. The acronym has been variously interpreted, perhaps flatteringly, but incorrectly, as Luria broth, Lennox broth, or Luria Bertani medium. For the historical record, the abbreviation LB was intended to stand for "lysogeny broth." (5, page 598).

[edit] Preparation

The following is a common method for the preparation of 1 litre of LB:

  • Measure out the following:
    • 10g tryptone
    • 5g yeast extract
    • 10g NaCl
  • Suspend the solids in ~800ml of distilled or deionized water.
  • Add further distilled or deionized water, in a measuring cylinder to ensure accuracy, to make a total of 1 litre.
  • Autoclave at 121°C.
  • After cooling, swirl the flask to ensure mixing, and the LB is ready for use. Try your very best to keep it sterile!

[edit] Adjusting the pH

Prior to autoclaving, some labs adjust the pH of LB to 7.5 or 8 with sodium hydroxide. The downside of using sodium hydroxide is that the pH will not be buffered which means that the bacteria will rapidly change the pH as they grow. To get around this some labs prefer to adjust the pH with 5-10 mmol/l TRIS buffer, diluted from 1 mol/l TRIS stock at the desired pH. However, it is not absolutely necessary to adjust the pH for most situations.

Since the buffering with Tris will also be largely ineffective in the face of substantial bacterial growth, adjusting the pH of LB in this particular manner is usually unnecessary. As such, use of Tris in some broth recipes (especially when the culture will be stored at room temperature conditions for extended periods of time) may be considered a cargo cult type instruction.

[edit] See also

[edit] References

  • Anderson, E. H. (1946). Growth requirement of virus-resistant mutants of Escherichia coli strain B. Proc. Natl. Acad. Sci. USA 32:120-128. PMID 16588724
  • Bertani, G. (1951). Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacteriol. 62:293-300. PMID 14888646 PDF
  • Luria, S. E., and J. W. Burrous. (1957). Hybridization between Escherichia coli and Shigella. J. Bacteriol. 74:461-476. PMID 13475269 PDF
  • Lennox, E. S. (1955). Transduction of linked genetic characters of the host by bacteriophage P1. Virology. 1:190-206. PMID 13267987
  • Luria, S. E., J. N. Adams, and R. C. Ting. (1960). Transduction of lactose-utilizing ability among strain of E. coli and S. dysenteriae and the properties of the transducing phage particles. Virology. 12:348-390. PMID 13764402
  • Miller, J. H. (1972). Experiment in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
  • Sambrook, J., E. F. Fritsch, and T. Maniatis. (1989). Molecular cloning: a laboratory manual, 2nd edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
  • Bertani, G. (2004). Lysogeny at mid-twentieth century: P1, P2, and other experimental systems. J. Bacteriology. 186:595-600. PMID 14729683 doi:10.1128/JB.186.3.595-600.2004