LTEE Media Recipes
Jesus E Chavarria-Palma, Jeffrey E Barrick
Abstract
Growth media used by the long-term E. coli evolution experiment.
- TA: Tetrazolium Arabinose for distinguishing Ara- and Ara+ strains in most competition assays. Colonies of Ara- strains typically appear red on TA agar, while those of Ara+ strains appear white. TA plates are generally incubated at 37°C for 24 h.
- DM: The basic medium used for propagating the long-term lines is Davis Minimal broth supplemented with glucose at a concentration of 25 mg per L, which we refer to as DM25. This medium supports a stationary-phase density of about 5 x 107 cells per ml for the founding strain of E. coli B.
- MG: Same basic composition as for DM liquid medium, except that we: add agar (as solidifying agent), increase the sugar concentration (so that colonies are robust), and sometimes use arabinose (instead of glucose). Glucose is used to examine the colonies on the standard minimal medium.
Steps
DM: Davis-Mingioli
To prepare 1L of DM:
Weigh dry components:
a. 5.34g of 7gof
b. 2g of
c. 1g of
d. 0.5g of
Add distilled water to a final volume of 1L
Autoclave using liquid program. Sterilization times are based on total volume:
| A | B |
|---|---|
| 75-200 | 20 |
| 200-500 | 25 |
| 500-1000 | 30 |
| 1000-1500 | 35 |
| 1500-2000 | 40 |
| >2000 | 60 |
Sterilization time
After autoclaving add the following stock solutions:
a. 1mL of previously sterilized10Mass / % volume
b. 1mL of filtered sterilized 0.2Mass / % volume
If preparing DM-glucose, add this volume of10Mass / % volume , to get the final concentration desired:
| A | B | C | D | E |
|---|---|---|---|---|
| 5 ml | DM500 | 0.05% | 500 mg/L | 2.78 mM |
| 250 µl | DM25 | 0.0025% | 25 mg/L | 139 µM |
| 20 ml | DM2000 | 0.2% | 2000 mg/L | 11.1 mM |
| 2.5 ml | DM250 | 0.025% | 250 mg/L | 1.39 µM |
| 10 ml | DM1000 | 0.1% | 1000 mg/L | 5.55 mM |
| 1 ml | DM100 | 0.010% | 100 mg/L | 694 µM |
TA: Tetrazolium Arabinose
To prepare 1.5L of TA:
Prepare media base by combining in a 2L flask:
a. 15g of
b. 1.5g of
c. 7.5g of
d. 24g of
e. 1.5mL of
Add distilled water to 1.3L
Separately, prepare sugar solution by combining:
a. 15g of
b. 200mL of distilled water
Autoclave both solutions, media base and sugar solution from and separatley and according to sterilization table in .
Combine sterile solutions, media base and sugar solution for a total of 1.5L
Add 1.5mL of (filter sterilized and stored at 4°C )5Mass / % volume
MG: Minimal glucose
To prepare 1L of MG:
When making these plates it is necessary to prepare and autoclave the 3 main parts (salt solution, agar base, and sugar solution) separately . Compounds that inhibit growth are produced when agar and phosphate or phosphate and glucose are autoclaved together.
Prepare salt solution , combine:
a. 5.3g of
b. 2g of
c. 1g of
d. 0.5g of
e. 400mL of distilled water
Autoclave salt solution according to
Prepare agar base by combining:
a. 16g of
b. 1mL of
c. 400mL of distilled water
Autoclave agar base according to
Prepare sugar solution by combining:
a. 4g of
b. 200mL of distilled water
Autoclave sugar solution according to
After the three parts have been autoclaved, combine the contents of the three flasks together while they are still warm add the following stock solutions:
a. 1mL of 10Mass / % volume (separately autoclaved stock)
b. 1mL of 0.2Mass / % volume (filter sterilized stock)
