Luria-Bertani (LB) Medium Preparation
Introduction
Materials
- Consumables
- tryptone
- yeast extract (yeastolate)
- NaCl
- bacto agar (optional)
- NaOH
- selectable component (e.g., antibiotic(s)) (optional)
- Disposables
- weigh dishes/paper
- petri dishes (optional)
- Equipment
- media bottle
- pH meter
- autoclave
- balance
Procedure
Component Composition
A A | B B | |
1 1 | Final Volume (mL)* | 100 |
A A | B B | |
1 1 | Component | Weight (g) |
2 2 | tryptone | 1 |
3 3 | yeast extract | 0.5 |
4 4 | NaCl | 1 |
5 5 | bacto agar (optional) | 1.5 |
Preparation
- Add ~80% of the final volume of dH2O to a sterile media bottle.
Generally, the capacity of the bottle should be greater than (not equal to) the final volume of media to be prepared in order to prevent the media from boiling over during the autoclave step.
Ensure you have a volumetric flask measuring your desired final volume.
- Adjust the final volume in the table above and add the calculated amounts of tryptone, yeast extract, and NaCl to the dH2O.
CRITICALDo not add agar at this point if you are preparing solid medium.
- Seal the cap of the media bottle and shake until components are completely dissolved.
- Neutralize (pH = 7.0) the solution using NaOH.
Approximately 1mL of 1N NaOH should be expected.
- Transfer the solution to a sterile volumetric flask and adjust the final volume using dH2O, then return it to the media bottle.
Ensure the solution is well mixed before returning it to the media bottle.
- SOLID MEDIUM ONLY Add the appropriate amount of agar to the solution.
CRITICALBacto agar does not need to dissolve at this point.
Note: The density of agar is ~1g/mL, it should only increase the final volume by 1.5%, which is negligible.
- Apply autoclave tape to the bottle and autoclave on liquids cycle. Read step 8.
CRITICALLiquids cycle.
- If adding selectable components or preparing solid medium, begin warming a water bath to 55°C.
SOLID MEDIUM ONLY, begin labeling approximately 1 plate per 15mL of medium.
- LIQUID MEDIUM ONLY If you are not adding selectable components, go to step 14.
- Allow solution to cool to 55°C in water bath.
- If adding selectable components, add them at this point.
CRITICALEnsure solution is well mixed.
- SOLID MEDIUM ONLY Pour approximately 15mL into each labeled petri dish and allow to cool to room temperature.
CRITICALEnsure entire bottom of dish is covered.
- SOLID MEDIUM ONLY Once media has gelled, replace the dishes inverted into their respective sleeve.
CRITICALSolid media should always be stored/incubated in the inverted position.
- Store media at 4°C.
Comments
While preparing the LB liquid medium from LB agar, I am facing two problem (not at the same time)
1. Either my medium solidifies at room temperature.
2. or I am getting precipitates in my LB medium.
I have prepared it by dissolving 32g of LB agar in i L of water.
Please let me know if you have any comments or suggestions about this.
Thanks,
Sweta
This protocol is adapted from Sambrook & Russell for the preparation of Luria-Bertani medium. It describes preparation of both liquid and 1.5% solid agar media.
Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual, 3 edn. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. pp. A2.2