Nov 25, 2024
TB Media Preparation
- 1Walther Straub Institute of Pharmacology and Toxicology, LMU - Munich, TUM;
- 2Walther Straub Institute of Pharmacology and Toxicology, LMU - Munich;
- 3Walther-Straub Institute of Pharmacology and Toxicology, LMU - Munich
- Luisa F Jimenez-Soto: * Corresponding author (l.jimenez(at)lmu.de);
Protocol Citation: Christoph Meister, Franziska Julia Silgoner, Tanja Krueger, Luisa F Jimenez-Soto 2024. TB Media Preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl49mqjgo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 17, 2024
Last Modified: November 25, 2024
Protocol Integer ID: 110165
Keywords: TB media, TB broth, Terrific Broth, plasmid isolation, bacterial culture
Funders Acknowledgement:
BMBF
Grant ID: SSTDBB - 16DKWN136B
Disclaimer
No conflict of interest.
Abstract
Terrific Broth (TB) as described by Tartoff, K.D. and Hobbs, C.A. (1987) is a nutritionally rich medium, with higher concentrations of peptone, yeast extract, and glycerol as a carbon source (Thermo Scientific | Terrific Broth). It supports high cell titers (Wood et al. 2017), and subsequent yields for plasmid DNA (Wood et al. 2017). While it has been referenced often we could not find the original Tartoff, K.D. and Hobbs, C.A. (1987) publication. What we present here has been passed down by oral tradition. We had sadly no access to the original publication.
Here we describe the steps we take to prepare the two solutions, denoted here as solution 1 and 2. Each is prepared and autoclaved separately before being combined in a 1:10 ratio shortly before inoculation with bacteria and antibiotics. The first solution contains all nutritional components, while the second has buffer characteristics, probably allowing a constant pH during the growth of the bacteria.
Image Attribution
Exotoxins Lab (TM)
Guidelines
- As good microbiological practice, all containers used for components should be labeled with the name of the solution, the date of creation / aliquote, and initials of the person who did it, in order to ask questions if something is needed.
- If you have doubts about how to perform a step in the protocol, consult your supervisor or someone who has prepared the solution before.
- For any questions, feel free to email us (exotoxinslab_(at)_gmail.com). We are more than happy to help you.
Materials
Materials
- Peptone / Tryptone from CaseinCarl RothCatalog #8952
- GlycerolCarl RothCatalog #3783
- Distilled WaterContributed by users (dH2O)
- Yeast ExtractCarl RothCatalog #2363.1
Equipment
Laboratory bottles borosilicate glass
NAME
Glassware
TYPE
Simax
BRAND
FL15
SKU
LINK
Laboratory bottles made of clear borosilicate glass 3.3 With white imprinted graduation, completely autoclavable With attached blue screw cap made of PP and blue pouring ring made of PP, in acc. with ISO 4796-1 Height is with attached screw cap!
SPECIFICATIONS
Solution 1:
| A | B | C | D | |
| dH2O | 996 mL | 498 mL | 249 mL | |
| Glycerol | 4 mL | 2 mL | 1 mL | |
| Tryptone | 12 g | 6 g | 3 g | |
| Yeast extract | 24 g | 12 g | 6 g | |
| 1 L | 500 mL | 250 ML |
Materials solution 1 for 1 L, 0.5 L and 0.2
Solution 2:
| A | B | C | |
| 100 mL | final c (10x) | ||
| Kaliumdihydrogenphosphate (KH2PO4) | 2.32 g | 0.17 M | |
| Dikaliumhydrogenphosphate (K2HPO4) | 16.4 g | 0.72 M | |
| dH2O | 100 mL |
Materials solution 2 (10x) for 100 mL
Protocol materials
Peptone / Tryptone from CaseinCarl RothCatalog #8952
Materials, Step 2
Yeast ExtractCarl RothCatalog #2363.1
Materials, Step 3
Distilled Water
In Materials and 3 steps
GlycerolCarl RothCatalog #3783
Materials, Step 4
Safety warnings
The process of autoclaving requires the use of high temperatures and specialized equipment. Make sure you have been instructed in the use of the autoclave.
When handling recently autoclaved liquids, their temperatures can be above 60°C, and can cause severe burns. Follow the safety protocols of your institution and use always personal protective equipment.
Before start
- Be aware of your lab's safety protocols and check the attached Warnings (Guidelines and Warnings) to see what steps are necessary to protect yourself, such as protocol-appropriate personal protective gear like lab coat, gloves, and glasses.
- Make sure to read the whole protocol before starting and have all the materials at hand.
- Before using any machine, make sure you have been properly instructed in its use, and read the Standard Operating Procedures (SOP) with the safety information provided by your institution or supervisor.
Preparation solution 1
Preparation solution 1
For 1 L of solution pour about 500 mL of Distilled WaterContributed by users into a measuring cylinder
Add 12 g of Peptone / Tryptone from CaseinCarl RothCatalog #8952
Add 24 g of Yeast ExtractCarl RothCatalog #2363.1
Add 4 mL of GlycerolCarl RothCatalog #3783
Add a magnetic rod to the cylinder and mix on a magnetic stirrer, until fully dissolved
Pour an additional 500 mL Distilled WaterContributed by users for a final volume of 1 L
Evenly distribute 500 ml of the solution into 2x 1 L bottles / flasks (see Materials for details). To avoid the explosion of the bottles during autoclaving, leave the lid loose and secure it with autoclave tape.
Autoclave at 121 °C at (psi) for 15 - 20 min. Make sure you know how to use the autoclave before you use it.
Preparation of solution 2
Preparation of solution 2
For 100 mL of solution pour about 50 mL of Distilled WaterContributed by users into a measuring cylinder
Add 2.32 g KH2PO4
Add 16.4 g K2HPO4
Add a magnetic rod/fish to the cylinder, and mix on a magnetic stirrer, until fully dissolved
Pour the solution into a 200 ml bottle. To avoid the explosion of the bottles during autoclaving, leave the lid loose and secured with autoclave tape.
Autoclave at 121 °C at (psi) for 15 - 20 min. Make sure you know how to use the autoclave before you use it. Do not autoclave together with solution 1. The difference in amounts (200 ml vs 500 ml) will cause a overheating in solution 2, if the temperature sensor is evaluating the 500 ml volume temperature.
Combining the solutions
Combining the solutions
After the solutions are autoclaved, close the lids tight, and store them at room temperature. Just before use, add solution 2 to solution 1 for a final concentration of 10 % (v/v) of solution 2 .
Protocol references
Tartoff KD, Hobbs CA. Improved media for growing plasmid and cosmid clones. Bethesda Res Lab Focus. 1987;9:12.
Wood WN, Smith KD, Ream JA, Lewis LK. Enhancing yields of low and single copy number plasmid DNAs from Escherichia coli cells. J Microbiol Methods. 2017 Feb;133:46-51. doi: 10.1016/j.mimet.2016.12.016. Epub 2016 Dec 23. PMID: 28024984; PMCID: PMC5286560.
Acknowledgements
To Walther Mothes, for showing me (LFJS) the media as optimal solution for low copy plasmid extraction.