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 casein peptones
• Vitamins (including B vitamins)
• Trace elements (e.g. nitrogen, sulfur, magnesium)
• Minerals

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.

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)

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).

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!

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 superstitious procedure without much scientific merit.

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

Description

Lysogeny broth (LB), more commonly called Luria Broth,  agar plates are typically used as a growth substrate for the culture of bacteria (e.g., E. Coli). Selective growth compounds may also be added to the media, such as antibiotics. Individual microorganisms placed on the plate will grow into individual colonies, each a clone genetically identical to the individual ancestor organism (except for the low, unavoidable rate of mutation). Thus, the plate can be used either to estimate the concentration of organisms in a liquid culture or a suitable dilution of that culture, using a colony counter, or to generate genetically pure cultures from a mixed culture of genetically different organisms, using a technique known as streaking. In this technique, a drop of the culture on the end of a thin, sterile loop of wire is “streaked” across the surface of the agar leaving organisms behind, a higher number at the beginning of the streak and a lower number at the end. At some point during a successful “streak”, the number of organisms deposited will be such that distinct individual colonies will grow in that area which may be removed for further culturing, using another sterile loop.

Procedure

This recipie makes about 1 L of media, sufficient for 30 plates. Preparation time is approximately 2 hours.

1. To a flask of volume at least 2 L, add:

• 10 g Tryptone
• 5 g Yeast Extract
• 5 g NaCl
• 800 mL of distilled water

2. Stir the solution until everything is completely dissolved.
3. Add 400ul of 5N NaOH with stirring to adjust the pH.
4. Bring the liquid level up to to 1000 ml with distilled water.
5. Add 15g of granulated agar to the liquid and stir until the agar is dissolved (about 1 minute).
6. Remove the stir bar, cover the flask with aluminum foil and autoclave for 20 min using the liquid cycle.
7. Cool down the medium until it is cool enough to be held in the hands (about 40^oC).
8. While the media is cooling, spray and wipe the bench with 95% ethanol.
9. Open a bag of sterile 3″ empty plates and place them in stacks of 10 plates with the lids up. Save the bag for later storage of the plates.
10. Label the plates for proper identification:

• LB only – single vertical black band
• LB + Ampicillin – (optional black band) single vertical red band
• LB + Chloramphenicol – single vertical blue band
• LB + Kanamycin – (optional black band) single vertical green band

11. When the media has cooled, add the appropriate amount of antibiotic(s) to the medium and gently swirl to mix:

• 100ug/mL Ampicillin
• 34ug/mL Chloramphenicol
• 10ug/mL Kanamycin

12. At this point, you can pour the LB agar from the flask into a sterile 500-mL beaker for easier transfer onto the plates.
13. Sterilize the flask mouth by flame. If any bubbles are present in the agar, you can burst them passing the flame quickly over the LB agar solution.
14. Open the lid of the top plate and flame the beaker mouth, then pour the LB agar onto the plate until about half-way full.
15. The plates should stand at room temperature for a day before being bagged and stored. They may be used for experiments later the same day if required.
16. Store the plates upside down inside the bag, to prevent them from drying out, and store at 4^oC.