TITLE: LTEE Media Recipes
AUTHORS: Jesus E Chavarria-Palma, Jeffrey E Barrick

[DESCRIPTION]
 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]
SECTION: DM: Davis-Mingioli
1. To prepare 1 L of DM:

SECTION: DM: Davis-Mingioli
1.2. Add distilled water to a final volume of 1 L 
SECTION: TA: Tetrazolium Arabinose
2. To prepare 1.5 L  of TA:

SECTION: DM: Davis-Mingioli
1.3. Autoclave using liquid program. Sterilization times are based on total volume:

 

SECTION: DM: Davis-Mingioli
1.4. After autoclaving add the following stock solutions:
a. 1 mL of previously sterilized at  10 Mass / % volume 
b. 1 mL of filtered sterilized   at 0.2 Mass / % volume 
SECTION: DM: Davis-Mingioli
1.5. If preparing DM-glucose, add this volume of (separately autoclaved stock) at 10 Mass / % volume ,   to get the final concentration desired: 
 
 
 
125 µl   250 µl   1.25 ml   DM25   0.0025%   25 mg/L   139 µM     0.5 ml   1 ml   5 ml   DM100   0.010%   100 mg/L   694 µM     1.25 ml   2.5 ml   12.5 ml   DM250   0.025%   250 mg/L   1.39 µM     2.5 ml   5 ml   25 ml   DM500   0.05%   500 mg/L   2.78 mM     5 ml   10 ml   50 ml   DM1000   0.1%   1000 mg/L   5.55 mM     10 ml   20 ml   100 ml   DM2000   0.2%   2000 mg/L   11.1 mM



 

 


SECTION: TA: Tetrazolium Arabinose
2.2. Add distilled water to 1.3 L 
SECTION: TA: Tetrazolium Arabinose
2.3. Separately, prepare sugar solution by combining:
a. 15 g of   
b. 200 mL of distilled water

 

SECTION: TA: Tetrazolium Arabinose
2.4. Autoclave both solutions, media base and sugar solution from    and   separatley  and according to sterilization table in   . 

SECTION: TA: Tetrazolium Arabinose
2.5. Combine sterile solutions, media base and sugar solution for a total of 1.5 L  
SECTION: TA: Tetrazolium Arabinose
2.6. Add 1.5 mL of (filter sterilized and stored at 4 °C )  at 5 Mass / % volume 

SECTION: MG: Minimal glucose
3.1. 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.
SECTION: MG: Minimal glucose
3.2. 
Prepare salt solution, combine:
a. 5.3 g of   
b. 2 g of   
c. 1 g of   
d. 0.5 g of   
e. 400 mL  of distilled water
SECTION: MG: Minimal glucose
3.4. Prepare agar base by combining:
a. 16 g of   
b. 1 mL  of   
c. 400 mL  of distilled water
SECTION: DM: Davis-Mingioli
1.1. Weigh dry components:
a. 5.34 g of   or 7 gof     
b. 2 g of   
c. 1 g of   
d. 0.5 g of   
SECTION: TA: Tetrazolium Arabinose
2.1. Prepare media base by combining in a 2L flask:
a. 15 g of  
b. 1.5 g of   
c. 7.5 g of   
d. 24 g of   
e. 1.5 mL of   
SECTION: MG: Minimal glucose
3. To prepare 1 L  of MG: 

SECTION: MG: Minimal glucose
3.6. Prepare sugar solution by combining:
a. 4 g of   
b. 200 mL of distilled water
 

SECTION: MG: Minimal glucose
3.7. Autoclave sugar solution according to   
SECTION: MG: Minimal glucose
3.3. Autoclave salt solution according to   
SECTION: MG: Minimal glucose
3.5. Autoclave agar base according to   
SECTION: MG: Minimal glucose
3.8. 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. 1 mL of   at 10 Mass / % volume  (separately autoclaved stock)
b. 1 mL of   at 0.2 Mass / % volume (filter sterilized stock)