LTEE Media Recipes

Jesus E Chavarria-Palma, Zachary D Blount, Jeffrey E Barrick

Published: 2023-12-30 DOI: 10.17504/protocols.io.81wgbyr31vpk/v3

Long-Term Evolution Experiment

Abstract

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.

Note

(1) Ara⁻ clones will typically form microcolonies on MA that are at the limits of visual inspection owing to trace substrates present in agar. (2) Because MA contains citrate, Cit⁺ clones from the Ara−3 population will form visible colonies on MA even if they have an Ara⁻ phenotype. When isolating Ara⁺ revertant mutants or inspecting the Ara− phenotype of Cit+ clones, use of MA formulated without citrate is recommended.

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

DM-glucose: Davis-Mingioli liquid medium supplemented with glucose

To prepare 1L of DM, follow these steps.

Note

DM media are described here:

Citation

B. C. Carlton and B. J. Brown 1981 Gene mutation Manual of methods for general bacteriology. American Society for Microbiology, Washington, D.C.

The DM-glucose medium formulation used in the LTEE is further described here:

Citation

Richard E. Lenski, Michael R. Rose, Suzanne C. Simpson, Scott C. Tadler 1991 Long-term experimental evolution in Escherichia coli. I. Adaptation and divergence during 2,000 generations American Naturalist https://www.jstor.org/stable/2462549

There is a typo in the Lenski et al. paper. The actual amount of thiamine hydrochloride in this recipe and what has been used throughout the LTEE is 0.0002% (w/v), not the 0.000002% (w/v) stated in the paper.

Note

DM medium was developed by Bernard D. Davis (1916-1994) and Elizabeth S. Mingioli (1926-1997) at the U.S. Public Health Service Tuberculosis Research Laboratory at Cornell University for use in the isolation of auxotrophic mutants of E. coli using penicillin (Davis and Mingioli 1950). Developed independently by Davis and by Joshua Lederberg (1925-2008) and Norton Zinder (1928-2012), the "penicillin method" takes advantage of the fact that penicillin only kills actively growing cells. Addition of penicillin to a culture growing in a minimal medium selects for auxotrophic mutants that are not growing owing to the inability to synthesize one or more needed nutrients. DM was a refinement of the medium that Davis had originally used for the penicillin method. It included the addition of 0.5 mg/L of citrate as a standard part of the recipe because Davis had previously found that adding citrate increased the efficiency with which penicillin killed growing cells. The medium came to be commonly used in microbiology and molecular biology as the penicillin method spread in the community.Citrate was later established to improve iron availability for E. coli, which in turn improves growth in the medium and thus the killing efficiency of penicillin. Under neutral pH and oxic conditions, iron largely occurs as the insoluble ferric ion (Fe3+). Citrate chelates ferric ions to form a soluble complex of ferric dicitrate. E. coli is able to bind ferric di-citrate and import the iron from it. The concentration of citrate needed for this role is ~10 μM, far lower than the 1700 μM found in DM. The excess amount of citrate in DM is likely owing to 0.5 mg/L being a convenient concentration that Davis did not see a need for optimizing owing to E. coli's inability to grow aerobically on it. While E. coli can grow in DM formulated without citrate, that growth is inconsistent and highly variable, likely reflecting sensitivity to minor fluctuations in the dissolved iron content of the water used. It is therefore not recommended.The original citation for DM medium may be found here:

Citation

Bernard D. Davis, Elizabeth S. Mingioli 1950 Mutants of Escherichia coli requiring methionine or vitamin B-12 Journal of Bacteriology https://doi.org/10.1128/jb.60.1.17-28.1950

This note is drawn from the following:

Citation

Blount ZD 2016 A case study in evolutionary contingency Studies in history and philosophy of biological and biomedical sciences https://doi.org/10.1016/j.shpsc.2015.12.007

1.1.

Weigh dry components:

a. 5.34g of or 7gof

b. 2g of

c. 1g of

d. 0.5g of

1.2.

Add distilled water to a final volume of 1L

1.3.

Autoclave for 1 hour at 121°C and a pressure of 15 psi or higher.

1.4.

After autoclaving add the following stock solutions:

a. 1mL of previously autoclaved at 10Mass / % volume

b. 1mL of filtered sterilized at 0.2Mass / % volume

1.5.

If preparing DM-glucose, add this volume of (separately autoclaved stock) at 10Mass / % volume , to get the desired final concentration:

A	B	C	D	E
250 µL	DM25	0.0025%	25 mg/L	139 µM
1 mL	DM100	0.010%	100 mg/L	694 µM
2.5 mL	DM250	0.025%	250 mg/L	1.39 µM
5 mL	DM500	0.05%	500 mg/L	2.78 mM
10 mL	DM1000	0.1%	1000 mg/L	5.55 mM
20 mL	DM2000	0.2%	2000 mg/L	11.1 mM
0 mL	DM0	0.0%	0 mg/L	0 mM

Note

Remember: DMX = DM + X mg/L glucose. Glucose may no longer limit the final growth density above approximately DM1000.

Note

Final composition:Sodium (Na⁺) = 5.1millimolar (mM) Potassium (K⁺) = 75.8millimolar (mM) Ammonium (NH₄) = 15.2millimolar (mM) Magnesium (Mg²⁺) = 0.83millimolar (mM) Sulfate (SO₄^2-) = 8.41millimolar (mM) Phosphate (PO₄^3-) = 45.3millimolar (mM) Citrate = 1.7millimolar (mM) (In DM25) Glucose = 139micromolar (µM)

1.6.

DM-glucose medium is stable for more than a year at room temperature.

Citation

Sterile Saline

To prepare 1L of Sterile Saline (0.85% w/v), follow these steps.

2.1.

Add 8.5g of to a 2 L flask.

2.2.

Add distilled water to 1000mL

2.3.

Autoclave for 1 hour at 121°C and a pressure of 15 psi or higher.

Note

It's also possible to make 5L at a time by adding 42.5g ofSodium Chlorideto a 6 L flask.

2.4.

Sterile saline is stable for years at room temperature.

Citation

TA agar: Tetrazolium Arabinose agar

To prepare 1L of TA agar, follow these steps.

Note

Tetrazolium sugar agar recipe used in the LTEE is derived from (Levin, Stewart and Chao, 2015)

Citation

Bruce R. Levin, Frank M. Stewart and Lin Chao 1977 Resource-Limited Growth, Competition, and Predation: A Model and Experimental Studies with Bacteria and Bacteriophage The American Naturalist http://www.jstor.org/stable/2459975

Note

On TA, bacteria may grow on amino acids from the tryptone in the medium or on arabinose or whatever other sugar is provided. Cells that are unable to grow on the provided sugar grow on the amino acids, which produces ammonia and thus increases the pH. At alkaline pH, TTC is converted to the intensely red, insoluble dye, formazan, which is sequestered in a granule in the cell. Owing to this accumulation of formazan, bacteria that are unable to grow on the provided sugar thus form red colonies. By contrast, cells that can grow on the sugar do so preferentially. They do not form ammonia and thus do not cause conversion of TTC to formazan, causing them to form colonies that are white to pinkish in color. If incubated over long periods of time, however, colonies formed by sugar-using bacteria will turn more red. This occurs for two reasons. First, there is some small amount of growth on the amino acids, and so formazan still builds up, albeit slowly. Second, over time, the cells in the colony can exhaust the sugar and switch to growing more on the amino acids, resulting in greater accumulation of formazan. TTC is used in a wide variety of indicator media. Its use to distinguish between sugar-using and non-sugar using bacteria traces to Joshua Lederberg. In 1948, Lederberg described using it to distinguish wild type E. coli that were able to ferment glucose, maltose, or lactose from mutants that were not. TA is a logical modification of this original use.Lederberg's original note is here:

Citation

Joshua Lederberg 1948 Detection of fermentative variants with tetrazolium Journal of Bacteriology https://doi.org/10.1128%2Fjb.56.5.695-695.1948

More information on the biochemistry of tetrazolium indicator dye may be found here:

Citation

Nikki Turner, W. E. Sandine, P.R. Elliker, E. A. Day 1963 Use of tetrazolium dyes in an agar medium for differentiation of streptococcus lactis and streptococcus cremoris Journal of Dairy Science https://doi.org/10.3168/jds.S0022-0302(63)89059-1

3.1.

Prepare basal medium by combining in a 2 L flask into which a stir bar has been placed:

a. 1g of

b. 1g of

c. 5g of

d. 16g of

e. 1mL of

3.2.

Add distilled water to 800L

3.3.

Separately, prepare sugar solution by combining:

a. 10g of

b. 200mL of distilled water

Note

It is also possible to make indicator plates for other sugars such as rhamnose, maltose, lactose, etc..

3.4.

Autoclave both the basal medium and the sugar solution separately for 1h 0m 0s at 121ºC and 15 psi or higher.

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 1mL of (TTC) at 5Mass / % volume and sugar solution for a total of 1L. 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.

3.6.

Pour plates. 1L 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.

Citation

MG agar: Minimal glucose agar

To prepare 1L of MG agar, follow these steps.

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.

4.2.

Prepare basal salt solution by combining:

a. 5.3g of

b. 2g of

c. 1g of

d. 0.5g of

e. 400mL of distilled water

4.3.

Prepare agar solution by combining:

a. 16g of

b. 1mL of 5% (v/v)

c. 400mL of distilled water

4.4.

Prepare sugar solution by combining:

a. 4g of

b. 200mL of distilled water

Note

Any sugar of interest can be substituted for glucose.

4.5.

Autoclave basal salt, agar, and sugar solutions for 1h 0m 0s at 121°C and at least 15 psi.

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. 1mL of at 10Mass / % volume (separately autoclaved stock)

b. 1mL of at 0.2Mass / % volume (filter sterilized stock)

4.7.

Pour plates. 1L 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.

Citation

MA agar: Minimal Arabinose agar

To prepare 1L of MA agar, follow these steps.

5.1.

Follow the instructions for MG agar, with the exception of the substitution of

in the place of glucose.

Note

Cit+ evolved clones from the Ara−3 population will form visible colonies on MA owing to the presence of citrate. When selecting for Ara⁺ revertant mutants of Cit⁺ clones, use of MA agar with citrate excluded from the formulation is recommended.

5.2.

Pour plates. 1L 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.

Citation

MC agar: Minimal Citrate agar

To prepare 1L of MC agar, follow these steps.

6.1.

Follow the same steps as for making MG agar, but with the following modifications:

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 1L of distilled water between just these two flasks.
To the basal salts solution, you will add 4.5g of instead of 0.5 g of a sugar.

When done,

CC agar: Christensen Citrate agar

To prepare 1L of CC agar, follow these steps.

7.1.

In a 2 L flask into which has been placed a stir bar, combine the following:

a. 3g of

b. 0.2g of

c. 0.5g of

d. 0.1g of

e. 0.4g of

f. 1.0g of

g. 5.0g of

h. 0.08g g of

i. 0.012g g of

j. 15g of

k. 1L of distilled water

7.2.

Adjust pH to 6.7 with NaOH (1 N or 10 N).

7.3.

Autoclave solution for 1h 0m 0s at 121°C and at least 15 psi.

7.4.

Pour plates. 1L 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.

Note

Alternate Formulations: Modified Christensen's Citrate Agar (MCCA): This version is a simplified formulation that excludes some of the more trace components. It works just as well for detecting citrate-users. For MCCA, exclude the L-Cysteine hydrochloride, ammonium iron(III) citrate, and Sodium Thiosulfate, and substitute 0.5 g of Ammonium sulfate. Otherwise prepare exactly as for the original formulation.Christensen's Citrate Broth (CCB): Prepare exactly as with CCA, but exclude the agar. Using the formulation of MCCA, sans agar, also works.

Appendix: Stock solutions

This section describes in more detail how to make each the stock solutions required for the above recipes.

8.1.

10% (w/v) MgSO4 ₄

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

8.2.

0.2% (w/v) Thiamine

Add0.5g of to a 500 mL or 1 L graduated cylinder. Add distilled water to a final volume of 250mL . 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).

8.3.

10% (w/v) D-Glucose

Add 25g of to a 500 mL or 1 L graduated cylinder. Add distilled water to a final volume of 250mL . 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.

8.4.

5% (w/v) Triphenyltetrazolium chloride (TTC)

Add 5g of (TTC) to a 250 mL graduated cylinder. Add distilled water to a final volume of 100mL . 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.

8.5.

5% (v/v) Antifoam solution

Add 12.5mL of to 237.5mL 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.

LTEE Media Recipes

Abstract

Steps

DM-glucose: Davis-Mingioli liquid medium supplemented with glucose

Sterile Saline

TA agar: Tetrazolium Arabinose agar

MG agar: Minimal glucose agar

MA agar: Minimal Arabinose agar

MC agar: Minimal Citrate agar

CC agar: Christensen Citrate agar

Appendix: Stock solutions

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