Jul 18, 2024
Version 4
LTEE Media Recipes V.4
Forked from a private protocol
- 1The University of Texas at Austin;
- 2Michigan State University
External link: https://the-ltee.org
Protocol Citation: Jesus E Chavarria-Palma, Zachary D Blount, Jeffrey E Barrick 2024. LTEE Media Recipes . protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbyr31vpk/v4Version created by Jeffrey E Barrick
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: December 07, 2023
Last Modified: July 18, 2024
Protocol Integer ID: 103664
Keywords: Long-Term Evolution Experiment, Experimental Evolution, Escherichia coli
Funders Acknowledgement:
National Science Foundation
Grant ID: DEB-1951307
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.
Protocol materials
Trisodium citrate dihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S1804
In 3 steps
Magnesium SulfateP212121
In 3 steps
L-( )-ArabinoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #A3256-500G
In 2 steps
AgarMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1296
In 3 steps
Antifoam B EmulsionMerck MilliporeSigma (Sigma-Aldrich)Catalog #A5757-250ML
In 3 steps
Phenol RedMerck MilliporeSigma (Sigma-Aldrich)Catalog #P3532-25G
Step 7.1
Potassium phosphate dibasic trihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9666
Step 1.1
Thiamine HClP212121
In 2 steps
235-Triphenyltetrazolium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #T8877
In 2 steps
Sodium ChlorideFisher ScientificCatalog # MK-7581-212
In 4 steps
Bacto™ Yeast ExtractThermo FisherCatalog #212750
In 2 steps
Ammonium sulfateCatalog #97061-184
In 2 steps
GlucoseP212121Catalog #Glucose
In 4 steps
L-Cysteine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #C1276
Step 7.1
Sodium ThiosulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #72049-250G
Step 7.1
Potassium phosphate (monobasic)P212121
In 3 steps
ammonium iron(III) citrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #F5879-100G
Step 7.1
Bacto™ TryptoneThermo FisherCatalog #211705
Step 3.1
Potassium phosphate (dibasic)P212121
In 2 steps
DM-glucose: Davis-Mingioli liquid medium supplemented with glucose
DM-glucose: Davis-Mingioli liquid medium supplemented with glucose
To prepare 1 L of DM, follow these steps.
Note
DM media are described here:
CITATION
The DM-glucose medium formulation used in the LTEE is further described here:
CITATION
NOTE: 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 History and Notes on Citrate
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
This note is drawn from the following:
CITATION
Weigh dry components:
a. 5.34 g of Potassium phosphate (dibasic)P212121 or 7 g of Potassium phosphate dibasic trihydrateMerck Millipore SigmaCatalog #P9666
b. 2 g of Potassium phosphate (monobasic)P212121
c. 1 g of Ammonium sulfateCatalog #97061-184
d. 0.5 g of Trisodium citrate dihydrateSigma-aldrichCatalog #S1804
Add distilled water to a final volume of 1 L
Autoclave for 1 hour at 121°C and a pressure of 15 psi or higher.
After autoclaving add the following stock solutions:
a. 1 mL of previously autoclaved Magnesium SulfateP212121 at 10 Mass / % volume s
b. 1 mL of filtered sterilized Thiamine HClP212121 at 0.2 Mass / % volume
If preparing DM-glucose, add this volume of GlucoseP212121Catalog #Glucose (separately autoclaved stock) at 10 Mass / % volume , to get the desired final concentration:
| per 1L of DM | DMX | [Glucose] (w/v) | [Glucose] (mg/L) | [Glucose] (M) | |
| 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+) = 5.1 millimolar (mM)
- Potassium (K+) = 75.8 millimolar (mM)
- Ammonium (NH4) = 15.2 millimolar (mM)
- Magnesium (Mg2+) = 0.83 millimolar (mM)
- Sulfate (SO42-) = 8.41 millimolar (mM)
- Phosphate (PO43-) = 45.3 millimolar (mM)
- Citrate = 1.7 millimolar (mM)
- (In DM25) Glucose = 139 micromolar (µM)
DM-glucose medium is stable for more than a year at room temperature.
Expected result
Bottle of DM25
Sterile Saline
Sterile Saline
To prepare 1 L of Sterile Saline (0.85% w/v), follow these steps.
Add 8.5 g of Sodium ChlorideFisher ScientificCatalog # MK-7581-212 to a 2 L flask.
Add distilled water to 1000 mL
Autoclave for 1 hour at 121°C and a pressure of 15 psi or higher.
Note
It's also possible to make 5 L at a time by adding 42.5 g ofSodium ChlorideFisher ScientificCatalog # MK-7581-212 to a 6 L flask.
Sterile saline is stable for years at room temperature.
Expected result
Bottle of Sterile Saline
TA agar: Tetrazolium Arabinose agar
TA agar: Tetrazolium Arabinose agar
To prepare 1 L of TA agar, follow these steps.
Note
Tetrazolium sugar agar recipe used in the LTEE is derived from (Levin, Stewart and Chao, 2015)
CITATION
Note
How TA Works
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.
TA Origins
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
More information on the biochemistry of tetrazolium indicator dye may be found here:
CITATION
Prepare basal medium by combining in a 2 L flask into which a stir bar has been placed:
a. 10 g of Bacto™ TryptoneThermo FisherCatalog #211705
b. 1 g of Bacto™ Yeast ExtractThermo FisherCatalog #212750
c. 5 g of Sodium ChlorideFisher ScientificCatalog # MK-7581-212
d. 16 g of AgarSigma-aldrichCatalog #A1296
e. 1 mL of Antifoam B EmulsionSigma-aldrichCatalog #A5757-250ML
Add distilled water to 800 L
Separately, prepare sugar solution by combining:
a. 10 g of L-( )-ArabinoseSigma-aldrichCatalog #A3256-500G
b. 200 mL of distilled water
Note
It is also possible to make indicator plates for other sugars such as rhamnose, maltose, lactose, etc..
Autoclave both the basal medium and the sugar solution separately for 01:00:00 at 121ºC and 15 psi or higher.
1h
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 235-Triphenyltetrazolium chlorideSigma-aldrichCatalog #T8877 (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.
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.
Expected result
TA Agar Plates
MG agar: Minimal glucose agar
MG agar: Minimal glucose agar
To prepare 1 L of MG agar, follow these steps.
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.
Prepare basal salt solution by combining:
a. 5.3 g of Potassium phosphate (dibasic)P212121
b. 2 g of Potassium phosphate (monobasic)P212121
c. 1 g of Ammonium sulfateCatalog #97061-184
d. 0.5 g of Trisodium citrate dihydrateSigma-aldrichCatalog #S1804
e. 400 mL of distilled water
Prepare agar solution by combining:
a. 16 g of AgarSigma-aldrichCatalog #A1296
b. 1 mL of 5 % (v/v) Antifoam B EmulsionSigma-aldrichCatalog #A5757-250ML
c. 400 mL of distilled water
Prepare sugar solution by combining:
a. 4 g of GlucoseP212121Catalog #Glucose
b. 200 mL of distilled water
Note
Any sugar of interest can be substituted for glucose.
Autoclave basal salt, agar, and sugar solutions for 01:00:00 at 121°C and at least 15 psi.
1h
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 Magnesium SulfateP212121 at 10 Mass / % volume (separately autoclaved stock)
b. 1 mL of Thiamine HClP212121 at 0.2 Mass / % volume (filter sterilized stock)
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.
Expected result
MG Agar Plates
MA agar: Minimal Arabinose agar
MA agar: Minimal Arabinose agar
To prepare 1 L of MA agar, follow these steps.
Follow the instructions for MG agar, with the exception of the substitution of L-( )-ArabinoseSigma-aldrichCatalog #A3256-500G
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.
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.
Expected result
MA Agar Plates
MC agar: Minimal Citrate agar
MC agar: Minimal Citrate agar
To prepare 1 L of MC agar, follow these steps.
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 Trisodium citrate dihydrateSigma-aldrichCatalog #S1804 instead of 0.5 g of a sugar.
When done,
CC agar: Christensen Citrate agar
CC agar: Christensen Citrate agar
1h
To prepare 1 L of CC agar, follow these steps.
In a 2 L flask into which has been placed a stir bar, combine the following:
a. 3 g of Sodium ChlorideFisher ScientificCatalog # MK-7581-212
b. 0.2 g of GlucoseP212121Catalog #Glucose
c. 0.5 g of Bacto™ Yeast ExtractThermo FisherCatalog #212750
d. 0.1 g of L-Cysteine hydrochlorideSigma AldrichCatalog #C1276
e. 0.4 g of ammonium iron(III) citrateSigma AldrichCatalog #F5879-100G
f. 1.0 g of Potassium phosphate (monobasic)P212121
g. 5.0 g of Sodium ChlorideFisher ScientificCatalog # MK-7581-212
h. 0.08 g g of Sodium ThiosulfateSigma AldrichCatalog #72049-250G
i. 0.012 g g of Phenol RedSigma AldrichCatalog #P3532-25G
j. 15 g of AgarSigma-aldrichCatalog #A1296
k. 1 L of distilled water
Adjust pH to 6.7 with NaOH (1 N or 10 N).
Autoclave solution for 01:00:00 at 121°C and at least 15 psi.
1h
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.
Christensen's Citrate Agar Plates
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 hydrochlorideSigma AldrichCatalog #C1276 , ammonium iron(III) citrateSigma AldrichCatalog #F5879-100G , and Sodium ThiosulfateSigma AldrichCatalog #72049-250G , and substitute 0.5 g of Ammonium sulfateCatalog #97061-184 . 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
Appendix: Stock solutions
This section describes in more detail how to make each the stock solutions required for the above recipes.
10% (w/v) MgSO4
Add 25 g of Magnesium SulfateP212121 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 .
0.2% (w/v) Thiamine
Add0.5 g of Thiamine HClP212121 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).
10% (w/v) D-Glucose
Add 25 g of GlucoseP212121Catalog #Glucose 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.
5% (w/v) Triphenyltetrazolium chloride (TTC)
Add 5 g of 235-Triphenyltetrazolium chlorideSigma-aldrichCatalog #T8877 (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.
5% (v/v) Antifoam solution
Add 12.5 mL of Antifoam B EmulsionSigma-aldrichCatalog #A5757-250ML 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.