Media Recipes

Liquid Media

Sterile Saline

Purpose: Used make dilutions of viable cells for plating or transfer to new media.

This saline concentration of 0.85% w/v (145 mM) is suitable for diluting E. coli before transferring to new media or plating on agar.

1L 5L Component
8.5 g 42.5 g NaCl

Add dH2O to final volume. Autoclave before use. Prepare large batches in 6 L flask and then aliquot out into bottles. It is not important to measure out exact volumes when dividing a large batch among bottles.

Included topic: ProtocolsRecipesSaline

LB: Lysogeny Broth / Luria-Bertani Medium (Miller)

Rich media used for routine culture of E. coli and other bacteria at high cell densities.

1L 5L Component
10 g 50 g Tryptone
5 g 25 g Yeast Extract
10 g 50 g NaCl

Add dH2O to final volume. Autoclave. If mixing up large batches and aliquoting, be sure to add exact volumes to final media bottles so that they will be ready for addition of antibiotic to known concentrations.

This recipe is from Miller JH. (1992) A Short Course in Bacterial Genetics. CSHL Press. Handbook Unit 25.5.

Note: Be aware that (1) there are several other formulations that may be called LB but vary the amount of NaCl. Using the wrong one can cause large changes in growth. (2) LB was originally supposed to stand for "Lysogeny Broth" and you may also see it called "Luria Broth" (more about this).

Expected results: E. coli strains grow to 5×109 cells/ml final density in LB.

Variant: 0.1×LB is made with 1 g Tryptone, 0.5 g Yeast Extract, and 10 g NaCl per liter. (It is 0.1× in the nutrients, but not the salt!)

Included topic: ProtocolsRecipesLysogenyBroth

DM: Davis-Mingioli

Growth medium used by the long-term E. coli evolution experiment.

1L 5L Component MW
5.3 g 26.7 g Potassium Phosphate (dibasic) K2HPO4 MW 174.18
2 g 10 g Potassium Phosphate (monobasic) KH2PO4 MW 136.07
1 g 5 g Ammonium Sulfate (NH4)2 SO4 MW 132.08
0.5 g 2.5 g Sodium Citrate (trisodium, dihydrate) Na3C6H5O7 (H2O)2 MW 294.10
Add dH2O to final volume and autoclave.

Note: if using Potassium phosphate (dibasic) trihydrate use 7g per L

After autoclaving add the following stock solutions:

1L 5L Component
1.0 ml 5 ml 10% (w/v) Magnesium Sulfate MgSO4 (separately autoclaved stock)
1.0 ml 5 ml 0.2% (w/v) Thiamine (vitamin B1) (filter sterilized)

And supplement with a carbon source.

If preparing DM-glucose (MW 180.16 g/mol), add this volume of 10% glucose solution (separately autoclaved stock) to get the final concentration desired:

1L 5L DMX [glucose] (w/v) [glucose] (mg/L) [glucose] (M)
250l 1.25 ml DM25 0.0025% 25 mg/L 139 M
1 ml 5 ml DM100 0.010% 100 mg/L 694 M
2.5 ml 12.5 ml DM250 0.025% 250 mg/L 1.39 M
5 ml 25 ml DM500 0.05% 500 mg/L 2.78 mM
10 ml 50 ml DM1000 0.1% 1000 mg/L 5.55 mM
20 ml 100 ml DM2000 0.2% 2000 mg/L 11.1 mM

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

Final composition
5.1 mM Sodium (Na+)
75.8 mM Potassium (K+)
15.2 mM Ammonium (NH4)
0.83 mM Magnesium (Mg2+)
8.41 mM Sulfate (SO42-)
45.3 mM Phosphate (PO43-)
1.70 mM Citrate
139 M Glucose (in DM25)

Included topic: ProtocolsRecipesDavisMingioli

NZY+ Broth

1L Component
10 g NZ Amine (casein hydrolysate)
5 g Yeast Extract
5 g NaCl

Add dH2O to final volume. Adjust pH to 7.5 using NaOH (Requires ~350 l of 10 N). Autoclave. Then add.

1L Component
12.5 ml 1 M MgCl2
12.5 ml / 15.0 ml 1 M / 10% (w/v) MgSO4 FW = 120.3 g/mol
40 ml 10% (w/v) glucose

Included topic: ProtocolsRecipesNZY+Broth

M9 Minimal Media

1L Component
6 g Sodium phosphate dibasic (anhydrous), Na2HPO4
3 g Potassium phosphate monobasic, KH2PO4
0.5 g Sodium chloride, NaCl
1 g Ammonium chloride, NH4Cl
Add dH2O to 1 liter. Autoclave.

After media is autoclaved add the following sterile ingredients:

1L Component
1 ml 1M Magnesium sulfate, MgSO4
1 ml 0.1M Calcium chloride CaCl2
10 ml 10% (w/v) glucose (or other carbon source)

Final composition
93.0 mM Sodium (Na+)
22.1 mM Potassium (K+)
18.7 mM Ammonium (NH4)
1.0 mM Calcium (Ca2+)
0.1 mM Magnesium (Mg2+)
29.2 mM Chloride (Cl-
0.1 mM Sulfate (SO42-)
42.2 mM Phosphate (PO43-)

Included topic: ProtocolsRecipesM9Minimal

R2A

R2A is a medium that can be used to grow a wide variety of soil microbes.

1L 5L Component
0.5 g 2.5 g Yeast Extract
0.5 g 2.5 g Proteose Peptone No. 3
0.5 g 2.5 g Casamino Acids
0.5 g 2.5 g Dextrose
0.5 g 2.5 g Soluble Starch
0.3 g 1.5 g Sodium Pyruvate
0.3 g 1.5 g Dipotassium Phosphate
0.05 g 0.25 g Magnesium Sulfate
Add dH2O to final volume and autoclave.

If making R2A solid media also add 15 g of agar and 1 ml of 5% antifoam per liter.

Included topic: ProtocolsRecipesR2A

RCV Medium

RCV is a complex media consisting of 3 different sub-componets that must be prepared ahead of time if they are not already made.

Main recipe

1L Component
10 mL 10% (NH4)2 SO4
40 mL 10% DL-malate, pH 6.8
50 mL Super salts (see below)
15 mL 0.64 KPO4, pH 6.8 (see below)

add 3/4 H2O before adding the KPO4
adjust pH to 6.8 if needed
if making plates add 16 g Agar per L

0.64M KPO4, pH 6.8

500mL Component
20 g KH2PO4
30 g K2HPO4

Super salts

1L Component
40 mL 1.0% EDTA
20 mL 20% MgSO4 5H2O
20 mL 7.5% CaCl2 2H2O
20 mL Trace elements (see below)
48 mL 0.5% FeSO4 7H2O
20 mL 0.1% thiamine-HCl

Trace Elements

250mL Component
0.3975 g MnSO4H2O
0.7 g H3BO3 (Boric Acid)
0.01 g Cu(NO3)2 3H2O
0.06 g ZnSO4 7H2O
0.1875 g NaMoO4 2H2O (Sodium molybdate VI dihydrate)

Included topic: ProtocolsRecipesRCVMedium

SOB: Super Optimal Broth

1L Final [ ] Component
5 g 0.5% yeast extract
20 g 2.0% tryptone
0.5 g 10 mM NaCl
0.186 g 2 mM KCl
2.4 g 20 mM MgSO4

Add 800 ml of dH2O and dissolve components. Adjust to pH 7.5 prior to use with 1 M NaOH (approximately 25 ml). Add dH2O to a final volume of 960 ml. Autoclave.

Included topic: ProtocolsRecipesSOB

SOC: Super Optimal Broth with Glucose

Used for recovery of cells after electroporation or heat shock transformation.

1L Final [ ] Component
5 g 0.5% yeast extract
20 g 2.0% tryptone
0.5 g 10 mM NaCl
0.186 g 2 mM KCl
2.4 g 20 mM MgSO4

Add 800 ml of water and dissolve components. Adjust to pH 7.5 prior to use with 1 M NaOH (approximately 25 ml). Add H2O to a final volume of 960 ml. Autoclave. When solution has cooled to 50C (cool enough to touch with bare hands for a few seconds), add 40 ml of autoclaved 10% glucose.

Included topic: ProtocolsRecipesSOC

TGY Medium

1L 5L Component
5 g 25 g Pancreatic digest of casein
5 g 25 g Yeast Extract
1g 5 g Glucose
1 g 5 g K2HPO4
Add dH2O to final volume and autoclave.

Included topic:ProtocolsRecipesTGY

DR: Defined minimal media for D. radiodurans

250 ml 500 ml Component
50 ml 100 ml 5x M9 salts
250 ul 500 ul 5mM MnCl2
250 ul 500 ul 0.8M MgCl2
250 ul 500 ul 0.18 M CaCl2
2.5 ml 5 ml Syringe filtered Vitamins Mix
500 ul 1 ml Syringe filtered Amino Acid mix (see below)
Add dH2O to final volume.
Note: All components are either pre-sterilized or syringe filtered so the only things that need to be autoclaved are the receiving bottle and the added water.

To prepare the amino acids:

Amount Amino Acid
1 g L-Cys
0.5 g L-His
0.5 g L-Met
Dissolve these amino acids in 40 ml of dH2O

Included topic:ProtocolsRecipesDR

YPS Medium

1L 1.5L Component
3.0 g 4.5 g Yeast Extract
3.0 g 4.5 g Peptone
2.0 mL 3.0 mL 1 M Magnesium sulfate
2.0 mL 3.0 mL 1 M Calcium chloride
add water to final volume, adjust pH to 7.0 autoclave

Included topic:ProtocolsRecipesYPSMedium

1416: 4-hydroxybenzoic acid medium (for JJ-1b, Bacillus sp.)

1L 4L Component
4.25 g 17.0 g Potassium Phosphate (dibasic) K2HPO4 trihydrate
1.00 g 4.00 g Sodium Phosphate (monobasic) NaH2PO4 monohydrate
2.00 g 8.00 g Ammonium chloride NH4Cl
0.20 g 0.40 g Magnesium Sulfate MgSO4 heptahydrate
12.0 mg 48.0 mg Ferrous Sulfate FeSO4 heptahydrate
3.00 mg 12.0 mg Manganese Sulfate MnSO4 monohydrate
3.00 mg 12.0 mg Zinc Sulfate ZnSO4 heptahydrate
1.00 mg 4.00 mg Cobalt (II) Sulfate CoSO4
0.10 g 0.40 g Nitrilotriacetic acid *
1.00 g 4.00 g 4-Hydroxybenzoic acid *

* Dissolve these components in water made alkaline with concentrated NaOH before adding other components.

Adjust medium for final pH 7.2, +/- 0.2. Autoclave for at least 15 minutes.

If plates needed, add 15.0 g of agar per liter to the above recipe.

Included topic:ProtocolsRecipes1416,4-hydroxybenzoicacidmedium

Solid Media

LB: Lysogeny Broth / Luria-Bertani (Miller) Agar

Combine in a 2 L flask the following:

1.5L Component
15 g Tryptone
7.5 g Yeast Extract
15 g NaCl
24 g Agar
1.5 ml 5% Antifoam

Add dH2O to 1.5 L. Autoclave.

Notes and variations:

  • Common synonyms are Luria-Broth and Luria-Bertani medium.
  • This is the "Miller" formulation of LB. Another common formulation has only 5 g NaCl per liter! They are NOT interchangeable.
  • If you are performing sacB counter-selection, you may need to use even lower salt concentrations.
  • To make "soft" agar for phage studies, use only 7 g agar per liter.

Included topic: ProtocolsRecipesLysogenyBrothAgar

TA: Tetrazolium Sugar (TA, TM, TL, ...)

Combine in a 2 L flask:

1.5L Component
15 g Tryptone
1.5 g Yeast Extract
7.5 g NaCl
24 g Agar
1.5 ml 5% Antifoam
Add dH2O to 1.3 liter.

Combine in a 500 ml flask:

1.5L Component
15 g Sugar (arabinose = TA, maltose = TM, lactose = TL)
Add dH2O to 0.2 liter.

Autoclave sugar and media separately. Total combined volume is 1.5 liters.

Combine and add:

1.5L Component
1.5 ml TTC (5%)

Source: Lenski Lab Protocol

Included topic: ProtocolsRecipesTetrazoliumSugar

MG: Minimal Glucose agar, aka DM: Davis Mingioli agar

Note: When making these plates it is necessary to prepare and autoclave the 3 main parts (salts, agar, and sugar) separately. Compounds that inhibit growth are produced when agar and phosphate or phosphate and glucose are autoclaved together.

1L 1.5L Component MW
5.3 g 8 g Potassium Phosphate (dibasic) K2HPO4 MW 174.18
2 g 3 g Potassium Phosphate (monobasic) KH2PO4 MW 136.07
1 g 1.5 g Ammonium Sulfate (NH4)2 SO4 MW 132.08
0.5 g 0.75 g Sodium Citrate (trisodium, dihydrate) Na3C6H5O7 (H2O)2 MW 294.10
333 ml 500 ml dH2O  
Autoclave.

Next, prepare the agar:

1L 1.5L Component
16 g 24 g Agar
1 ml 1.5 ml Antifoam (5%)
333 ml 500 ml dH2O
Autoclave.

Next, prepare the sugar (0.2%):

1L 1.5L Component
4 g 6 g Glucose (or other sugar)
333 ml 500 ml dH2O
Autoclave.

After the 3 parts have been autoclaved, combine the contents of the three flasks together while they are still warm add the following stock solutions:

1L 1.5L Component
1.0 ml 1.5 ml 10% Magnesium Sulfate MgSO4 (separately autoclaved stock)
1.0 ml 1.5 ml 0.2% Thiamin (vitamin B1) (filter sterilized stock)

Source: Lenski Lab Protocol

Included topic: ProtocolsRecipesMinimalGlucose

MC: Minimal Citrate

Prepared the same as MG: Minimal Glucose with the following changes:

  • No glucose
  • 4.5 g/L Sodium Citrate (trisodium, dihydrate)

Included topic: ProtocolsRecipesMinimalCitrate

M9 Minimal Media Plates

1L Component
6 g Sodium phosphate, Na2HPO4H2O
3 g Potassium phosphate, KH2PO4
0.5 g Sodium chloride, NaCl
1 g Ammonium chloride, NH4Cl
16 g Agar
1 ml Antifoam
Add dH2O to 1 liter. Autoclave.

After media is autoclaved add the following sterile ingredients:

1L Component
1 ml 1M Magnesium sulfate, MgSO4
1 ml 0.1M Calcium chloride CaCl2
10 ml 10% (w/v) glucose (or other carbon source)

Included topic: ProtocolsRecipesM9

SOB: Super Optimal Broth Plates

1L Final [ ] Component
5 g 0.5% yeast extract
20 g 2.0% tryptone
0.5 g 10 mM NaCl
0.186 g 2 mM KCl
2.4 g 20 mM MgSO4
16 g   Agar
1ml   Antifoam

Add 800 ml of water and dissolve components. Adjust to pH 7.5 prior to use with 1 M NaOH (approximately 25 ml). Add H2O to a final volume of 960 ml. Autoclave.

Included topic: ProtocolsRecipesSOBplates

PA: Lac Papillation Agar

Make 10× Minimal A Salts.

10× Minimal A Salts
1L Component MW
80 g Potassium Phosphate (dibasic) K2HPO4 MW 174.18
45 g Potassium Phosphate (monobasic) KH2PO4 MW 136.07
10 g Ammonium Sulfate (NH4)2 SO4 MW 132.08
5 g Sodium Citrate (trisodium, dihydrate) Na3C6H5O7 (H2O)2 MW 294.10
to 1L dH2O  
Autoclave. Salt solution can be stored at room temperature.

Agar Solution
1L Component
15 g Agar
to 490 ml dH2O
Autoclave.

Sugar Solution
1L Component
2 g Glucose
to 400 ml dH2O
Autoclave.

After autoclaving, combine agar and sugar and add:

1L Component
100 ml 10× Minimal A Salts
6 ml 10% w/v (0.083M Mg2+) Magnesium Sulfate, anhydrous MgSO4 (autoclaved) MW 120.37
2.5 ml 0.2% w/v Thiamin (vitamin B1) (filter sterilized)
1 ml 500 mg/ml P-Gal (phenyl β-D-galactoside) (filter sterilized)
1 ml 40 mg/ml X-Gal (5-bromo-4-chloro-3-indolyl β-D-galactoside) (filter sterilized)

Note: The amounts added of some standard components (including agar) are slightly different for this recipe. Volumes have also been altered from the original Miller recipe to use the same stock solutions we have available for making DM plates. The final concentrations are 0.2% glucose, 0.05% P-Gal, and 0.004% X-Gal (w/v).

Sources:

  1. Nghiem, Y., Cabrera, M., Cupples, C.G., Miller, J.H. (1988) The mutY gene : A mutator locus in Escherichia coli that generates GC→TA transversions. Proc. Natl. Acad. Sci. U.S.A. 85:2709-2713.
  2. Miller J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Included topic: ProtocolsPapillationAgar

TGY Medium

1.5L Component
7.5 g Pancreatic digest of casein
7.5 g Yeast Extract
1.5g Glucose
1.5 g K2HPO4
24g Agar
1.5mL Antifoam

Add dH2O to final volume and autoclave.

Included topic: ProtocolsTGYPlates

Stab Agar

Making agar stabs for storage and transport of bacterial strains.

1L Component
10 g Tryptone
5 g Yeast extract
10 g Sodium chloride
6 g Agar

Autoclave. Transfer to cryovials before media cools. Can store the vials at 4 C for a few months.

Included topic: ProtocolsRecipesStabAgar

Special Media

Included topic: ProtocolsRecipesSpecialMedia

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