LB broth / LB medium

Giuseppe Bertani formulated LB broth in 1951 in an attempt to optimise Shigella growth and plaque formation. Although LB media is known as “Luria Broth”, “Luria-Bertani” medium, or “Lennox Broth”, it was initially named as “Lysogeny Broth”. LB broth’s agar form should be designated LA but now referring as LBA/LB. There are many formulations available for LB medium, in this article, we are providing most used formula (based on CSH protocols).

Applications

  • LB is a rich medium, used as general purpose bacterial culture medium especially Enterobacteriaceae members (E.coli is one among them).
  • Lysogeny broth is also used for coliphage plaque assays.

pH

Cold Spring Harbor Protocol recommends pH to be 7.0. However, depending on the application pH can be 7.0 – 8.0. Adjust the pH with 1N NaOH and 1N HCl. Although the protein/peptide component of the medium shows some degree of buffering capacity, which is not sufficient for rapidly growing bacterial population. For general purposes, this is not an issue. However, some labs prefer to make LB in 5-10mM TRIS buffer of the desired pH.

Composition

LB broth / LB medium
Adjust the pH to 7.0 with 1 N NaOH and 1N HCl.
LB broth / LB medium
Adjust the pH to 7.0 with 1 N NaOH and 1N HCl.
Reagent For 100 mL For 500 mL For 1000 mL
Tryptone 1 g 5 g 10 g
NaCl 1 g 5 g 10 g
Yeast extract 0.5 g 2.5 g 5 g
Agar 1.5 g 7.5 g 15 g

Reagents

Along with the reagents mentioned in the table 1N NaOH.
1N HCl.

  • 1N NaOH.
  • 1N HCl.

Materials and instruments

  • Glass Beaker.
  • Conical flasks.
  • Cotton plugs.
  • pH Meter.
  • Pipettes and tips.
  • Petri dish.
  • Weighing balance and paper boats.
  • Magnetic stirrer and pellet.

Procedure

  1. Determine the volume to be made and weigh the reagents accordingly (The precalculated values in the table for specific volume might be helpful).
  2. Add the reagents to a glass beaker and make up the volume to 90% of planned with ddH2O (e.g., 900 ml if you have planned for 1L broth). DO NOT add agar (if preparing LB medium with agar) at this point.
  3. Dissolve the components in the beaker using magnetic stirrer.
  4. Adjust the pH to the desired value. (see the pH section above for details).
  5. Adjust the broth to final volume using ddH2O.
  6. Transfer the broth to conical flask or aliquot into smaller volumes.
  7. Add agar if planned to make LB medium with agar. Adding agar after aliquoting will ensure equal distribution.
  8. Close the mouth of the flask with a cotton plug. Seal it further with paper and rubber band.
  9. Autoclave for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle.

Storage

Store broth at room temperature.

Caution

  • Opening and closing of media should be done in aseptic conditions only. Opening in the open air might lead to contamination.
  • Adding agar before adjusting pH might result in mild hydrolysis.

References

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