LB broth / LB medium
Introduction
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 the 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.
Composition
| 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 |
Table 1. Composition of LB agar.
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 the 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.
Preparation
- Determine the volume to be made and weigh the reagents accordingly (The precalculated values in the table for specific volume might be helpful).
- 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.
- Dissolve the components in the beaker using a magnetic stirrer.
- Adjust the pH to the desired value. (see the pH section above for details).
- Adjust the broth to final volume using ddH2O.
- Transfer the broth to conical flask or aliquot into smaller volumes.
- Add agar if planned to make LB medium with agar. Adding agar after aliquoting will ensure equal distribution.
- Close the mouth of the flask with a cotton plug. Seal it further with paper and rubber band.
- Autoclave for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle.
Storage
Store broth at room temperature.
Points to be noted
- Opening and closing of media should be done in aseptic conditions only.
- Adding agar before adjusting pH might result in mild hydrolysis.