Terrific Broth (TB) Medium Preparation
Introduction
Materials
- Consumables
- tryptone
- yeast extract (yeastolate)
- glycerol
- potassium phosphate (0.17M monobasic, 0.72M dibasic)
- bacto agar (optional)
- Disposables
- weigh dishes/paper
- petri dishes (optional)
- Equipment
- media bottle
- autoclave
- balance
Procedure
Component Composition
A A | B B | |
1 1 | Final Volume (mL)* | 1000 |
A A | |
1 1 | Autoclave Volume (mL) |
2 2 | 900 |
A A | B B | C C | |
1 1 | Component | Amount | Unit |
2 2 | tryptone | 12 | g |
3 3 | yeast extract | 24 | g |
4 4 | glycerol | 4 | mL |
5 5 | potassium phosphate (0.17M monobasic, 0.72 M dibasic) | 100 | mL |
6 6 | bacto agar (optional) | 15 | g |
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 autoclave volume.
- Adjust the final volume in the table above and add the calculated amounts of tryptone, yeast extract, and glycerol 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.
- Transfer the solution to a sterile volumetric flask and adjust the volume to the Autoclave 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.
- Begin warming a water bath to 55°C.
SOLID MEDIUM ONLY, begin labeling approximately 1 plate per 15mLof medium.
- Allow solution to cool to 55°C in water bath.
- Add the calculated amount of potassium phosphate solution (0.17M monobasic, 0.72M dibasic).
CRITICAL Ensure solution is well mixed.
- If adding selectable components, add them at this point.
CRITICAL Ensure solution is well mixed.
- SOLID MEDIUM ONLY Pour approximately 15mL into each labeled petri dish and allow to cool to room temperature.
CRITICAL Ensure entire bottom of dish is covered.
- SOLID MEDIUM ONLY Once media has gelled, replace the dishes inverted into their respective sleeve.
CRITICAL Solid media should always be stored/incubated in the inverted position.
- Store media at 4°C.
Comments
This protocol is adapted from Sambrook & Russell for the preparation of Terrific Broth (TB) 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. A2.4