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

[DESCRIPTION]
This protocol describes recipes to prepare growth media and reagents used in the E. coli long-term evolution experiment (LTEE). 

Section 1: DM-glucose, Davis-Mingioli liquid medium supplemented with glucose
Section 2: Sterile Saline
Section 3: TA agar, Tetrazolium Arabinose agar
Section 4: MG agar, Minimal Glucose agar (equivalent to DM agar)
Section 5: MA agar, Minimal Arabinose agar
Section 6: MC agar, Minimal Citrate agar
Section 7: CC agar, Christensen Citrate agar
Section 8: Stock Solutions, detailed instructions for stock solutions needed for media preparation

DM-glucose: Davis-Mingioli medium (or sometimes called Davis Minimal medium) supplemented with glucose is used for propagating the LTEE populations and for performing related experiments. For propagating the LTEE, glucose is added to a concentration of 25 mg/L, which we refer to as "DM25". DM25 supports a stationary-phase density of about 5×107 cells/mL for E. coli REL606 and REL607, the founding strains of the LTEE. (The stationary-phase density of evolved LTEE clones varies, but tends to be approximately half that of the ancestral strains.) DM with higher concentrations of glucose is used for reviving cells from freezer stocks or for growing many cells to harvest for certain experiments. These other DM formulations are named in an analogous fashion of DMX, where X is the concentration of glucose in mg/L (e.g., 1000 mg/L glucose in DM1000).

Sterile Saline: Used to dilute E. coli cultures, for instance when plating on agar to isolate colonies or to count CFUs to determine cell titers.

TA agar: Tetrazolium Arabinose agar plates are used for distinguishing E. coli cells that can grow on the sugar arabinose (Ara+) from those that cannot (Ara–). Plating dilutions that give 150-250 colonies are used for monitoring the LTEE for contamination and also for co-culture competition assays that measure the relative fitness of two strains. Colonies grown from Ara– cells appear red on TA agar, while those of Ara+ strains appear pinkish-white. These phenotypes are very clear after 24 hours of incubation at 37°C for the REL606 (Ara–) and REL607 (Ara+) ancestors of the LTEE. Colonies of evolved clones can exhibit a wide variation of these color phenotypes. Some evolved clones may take longer than 24 hours to form visible colonies on TA.

MG agar: Minimal Glucose agar has the same base composition as DM-glucose liquid medium, except agar is added as a solidifying agent, and the glucose concentration is increased to 4 g/L to support the growth of colonies. Plating dilutions that give 150-250 colonies or streaking out on MG-agar is used to isolate colonies from LTEE populations. Dilutions of the LTEE populations can also be plated on MG agar to monitor for unexpected growth, colony appearance, or CFU numbers that could indicate contamination. Ancestral clones form colonies within 24 hours on MG agar. Evolved clones typically also form colonies within 24 hours on MG agar, but some may take longer. Evolved clones also generally produce larger colonies than the ancestors on MG.

MA agar: Minimal Arabinose agar is the same as MG agar except that the sugar arabinose is used instead of glucose.  Ara– cells like those of strain REL606 will not form colonies on MA agar. Only Ara+ cells like those of strain REL607 will. Plating dilutions from the LTEE populations that give 150-250 CFUs on MA agar can be used to monitor the Ara– populations for contamination from Ara+ populations. The Ara+ ancestor and evolved strains generally form colonies within 24 hours on MA agar. However, some Ara+ populations have lost the ability to form colonies at later generations. Plating a large number of Ara– cells (>109) on an MA plate can also be used to select spontaneous mutants that have reverted from the Ara– marker state to the Ara+ marker state. Reversion to Ara+ among LTEE clones is usually via a single nucleotide substitution mutation in the araC gene. This mutation occurs at a rate of ~1010 cells/generation among non-mutator clones, and much higher among clones with mutator phenotypes.

 
MC agar: Minimal Citrate agar is the same as MG/MA agar except that citrate is used as the carbon source. Strains that have evolved citrate utilization (Cit+) can form colonies on MC agar.

CC agar: Christensen Citrate agar is an indicator medium that can be used to detect weak citrate utilization in colonies on the basis of a color change even, for strains that may not be able to form colonies on MC agar.

[STEPS]
SECTION: DM-glucose: Davis-Mingioli liquid medium supplemented with glucose
1. To prepare 1 L of DM, follow these steps.

 

 

SECTION: DM-glucose: Davis-Mingioli liquid medium supplemented with glucose
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: DM-glucose: Davis-Mingioli liquid medium supplemented with glucose
1.2. Add distilled water to a final volume of 1 L 
SECTION: DM-glucose: Davis-Mingioli liquid medium supplemented with glucose
1.3. Autoclave for 1 hour at 121°C and a pressure of 15 psi or higher. 

SECTION: DM-glucose: Davis-Mingioli liquid medium supplemented with glucose
1.4. After autoclaving add the following stock solutions:
a. 1 mL of previously autoclaved   at  10 Mass / % volume 
b. 1 mL of filtered sterilized   at 0.2 Mass / % volume 
SECTION: DM-glucose: Davis-Mingioli liquid medium supplemented with glucose
1.5. If preparing DM-glucose, add this volume of   (separately autoclaved stock) at 10 Mass / % volume ,   to get the desired final concentration: 
 
 
 
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 agar: Tetrazolium Arabinose agar
3. To prepare 1 L  of TA agar, follow these steps.


 

 


SECTION: TA agar: Tetrazolium Arabinose agar
3.1. Prepare basal medium by combining in a 2 L flask into which a stir bar has been placed:
a. 1 g of  
b. 1 g of   
c. 5 g of   
d. 16 g of   
e. 1 mL of   
SECTION: TA agar: Tetrazolium Arabinose agar
3.2. Add distilled water to 800 L 
SECTION: TA agar: Tetrazolium Arabinose agar
3.3. Separately, prepare sugar solution by combining:
a. 10 g of   
b. 200 mL of distilled water

 

SECTION: TA agar: Tetrazolium Arabinose agar
3.4. Autoclave both the basal medium and the sugar solution separately for 60 min at 121ºC and 15 psi or higher. 



SECTION: TA agar: Tetrazolium Arabinose agar
3.5. When the basal medium has cooled to the point at which its flask can be touched with the back of the hand for 5 second without pain, add 1 mL of   (TTC) at 5 Mass / % volume and sugar solution for a total of 1 L. Place flask with combined medium on a stir plate and stir at medium speed for 5 minutes to fully mix.  

Note: TTC stock should be filtered sterilized and stored at 4 °C. TTC is sensitive to light, so bottles of TTC stock solution should be stored in the dark and wrapped in foil to reduce light exposure. 
SECTION: MG agar: Minimal glucose agar
4. To prepare 1 L  of MG agar, follow these steps.

SECTION: MG agar: Minimal glucose agar
4.1. MG agar is composed of 3 solutions (basal salt solution, agar solution, and sugar solution), which must be prepared and autoclaved separately. If combined and autoclaved together, the components will react to produce compounds that will inhibit bacterial growth.
SECTION: MG agar: Minimal glucose agar
4.2. 
Prepare basal salt solution by combining:
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 agar: Minimal glucose agar
4.3. Prepare agar solution by combining:
a. 16 g of   
b. 1 mL  of 5 % (v/v)    
c. 400 mL  of distilled water
SECTION: MG agar: Minimal glucose agar
4.5. Autoclave basal salt, agar, and sugar solutions for 60 min at 121°C and at least 15 psi.

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

SECTION: MG agar: Minimal glucose agar
4.6. 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)

SECTION: MA agar: Minimal Arabinose agar
5. To prepare 1 L  of MA agar, follow these steps.
SECTION: TA agar: Tetrazolium Arabinose agar
3.6. Pour plates. 1 L will make approximately 35-45 plates. TA agar plates can be stored for at least one to two months at 4°C or one to two weeks at room temperature. While bacterial colonies will form on older medium, the TTC breaks down over time, reducing color differentiation between sugar− and sugar+ colonies. Exposure to light increases the rate at which TTC in the medium breaks down.

 

SECTION: MG agar: Minimal glucose agar
4.7. Pour plates. 1 L will make approximately 35 - 45 plates. MG agar plates can be stored for at least one to two months at 4°C or one to two weeks at room temperature.

 

SECTION: MA agar: Minimal Arabinose agar
5.1. Follow the instructions for MG agar, with the exception of the substitution of  
in the place of glucose.  

 

SECTION: Sterile Saline
2. To prepare 1 L  of Sterile Saline (0.85% w/v), follow these steps.
SECTION: Sterile Saline
2.1. Add 8.5 g of   to a 2 L flask.
SECTION: Sterile Saline
2.2. Add distilled water to 1000 mL 
SECTION: Sterile Saline
2.3. Autoclave for 1 hour at 121°C and a pressure of 15 psi or higher.


 

5.2. Pour plates. 1 L will make approximately 35 - 45 plates. MA agar plates can be stored for up to two months at 4°C or one to two weeks at room temperature.

 

SECTION: DM-glucose: Davis-Mingioli liquid medium supplemented with glucose
1.6. DM-glucose medium is stable for more than a year at room temperature.

 

SECTION: Sterile Saline
2.4. Sterile saline is stable for years at room temperature.

 

SECTION: MC agar: Minimal Citrate agar
6. To prepare 1 L  of MC agar, follow these steps.

SECTION: CC agar: Christensen Citrate agar
7. To prepare 1 L of CC agar, follow these steps.


SECTION: MC agar: Minimal Citrate agar
6.1. Follow the same steps as for making MG agar, but with the following modifications:

1. Do not prepare a sugar flask. You will thus have only two flasks: one for the basal salts solution and one for the agar solution. You will thus divide the 1 L of distilled water between just these two flasks.

2. To the basal salts solution, you will add 4.5 g of   instead of 0.5 g of a sugar.

When done,   

SECTION: CC agar: Christensen Citrate agar
7.1. In a 2 L flask into which has been placed a stir bar, combine the following:
a. 3 g of   
b. 0.2 g of   
c. 0.5 g of   
d. 0.1 g of   
e. 0.4 g of  
f. 1.0 g of   
g. 5.0 g of   
h. 0.08 g g of  
i. 0.012 g g of  
j. 15 g of   
k. 1 L of distilled water


SECTION: CC agar: Christensen Citrate agar
7.2. Adjust pH to 6.7 with NaOH (1 N or 10 N).

SECTION: CC agar: Christensen Citrate agar
7.3. Autoclave solution for 60 min at 121°C and at least 15 psi.

SECTION: CC agar: Christensen Citrate agar
7.4. Pour plates. 1 L will make approximately 35 - 45 plates. CC agar plates can be stored at room temperature for up to 4 months or at 4ºC for up to a year.

 
 


SECTION: Appendix: Stock solutions
8. This section describes in more detail how to make each the stock solutions required for the above recipes.

SECTION: Appendix: Stock solutions
8.5. 5% (v/v) Antifoam solution

Add 12.5 mL  of   to 237.5 mL of distilled water in a 250 mL glass bottle. Autoclave to sterilize.

Note: The reagent from Sigma-Aldrich comes as a 10% emulsion in water. This stock solution is 5% (v/v) of the reagent, so it is actually 0.5% v/v of  Antifoam B.
SECTION: Appendix: Stock solutions
8.1. 10% (w/v) MgSO4

Add 25 g of   to a 500 mL or 1 L graduated cylinder. Add distilled water to a final volume of 250 mL . Fully mix by swirling as you do so. Pour the solution into a 250 mL bottle. Autoclave to sterilize. Store at Room temperature .
SECTION: Appendix: Stock solutions
8.2. 0.2% (w/v) Thiamine

Add0.5 g of    to a 500 mL or 1 L graduated cylinder. Add distilled water to a final volume of  250 mL . Fully mix by swirling as you do so. Filter sterilize the solution into a pre-autoclaved 250 mL bottle. Store at 4 °C .

Note: This solution is 0.2% (w/v) in thiamine•HCl — NOT in thiamine. The concentration of thiamine is actually 89.2% of this or 0.178% (w/v) if you account for the fact that the reagent being dissolved is not pure thiamine. (The molecular weight of thiamine•HCl is 337.27, and the molecular weight of HCl is 36.46).
SECTION: Appendix: Stock solutions
8.3. 10% (w/v) D-Glucose

Add 25 g of   to a 500 mL or 1 L graduated cylinder. Add distilled water to a final volume of 250 mL . Fully mix by swirling as you do so. Pour solution into a 250 mL bottle. Autoclave to sterilize. Store at Room temperature .

Note: D-Dextrose is a synonym for D-glucose that you may find on many reagent containers. Typically, if D- is not specified in the labeling, you can assume that it is the correct D-sugar.
SECTION: Appendix: Stock solutions
8.4. 5% (w/v) Triphenyltetrazolium chloride (TTC) 

Add 5 g of   (TTC) to a 250 mL graduated cylinder. Add distilled water to a final volume of  100 mL . Fully mix by swirling as you do so. Filter sterilize the solution into a pre-autoclaved 100 mL bottle. Store at 4 °C. Wrap the bottle in foil. TTC is sensitive to light and can degrade over time.

Note: The final solution should be a pale yellow color.