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).
- 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.
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
|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|
Along with the reagents mentioned in the table 1N NaOH.
- 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.
- 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 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.
Store broth at room temperature.
- 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.
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