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

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Included topic: ProtocolsRecipesLuriaBertani

DM: Davis-Mingioli

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

0.5L	1L	4.4L	5L	Component	MW
2.67 g	5.34 g	23.32 g	26.7 g	Potassium Phosphate (dibasic) K2HPO4	MW 174.18
1 g	2 g	8 g	10 g	Potassium Phosphate (monobasic) KH2PO4	MW 136.07
0.5 g	1 g	4.4 g	5 g	Ammonium Sulfate (NH4)2 SO4	MW 132.08
0.25 g	0.5 g	2.2 g	2.5 g	Sodium Citrate (trisodium, dihydrate) Na3C6H5O7 (H2O)2	MW 294.10

Add dH₂O to final volume and autoclave.

Note: 4.4 L of DM makes 8 bottles with 550 ml each.

Note: If using Potassium phosphate (dibasic) trihydrate (MW 228.22) use 7g per L

After autoclaving add the following stock solutions:

0.5L	550mL	1L	5L	Component
0.5 ml	550 µl	1.0 ml	5 ml	10% (w/v) Magnesium Sulfate MgSO4 (separately autoclaved stock)
0.5 ml	1000 µl	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:

0.5L	550mL	1L	5L	DMX	[glucose] (w/v)	[glucose] (mg/L)	[glucose] (M)
125 µl	137.5 µl	250 µl	1.25 ml	DM25	0.0025%	25 mg/L	139 µM
0.5 ml	0.55 ml	1 ml	5 ml	DM100	0.010%	100 mg/L	694 µM
1.25 ml	1.375 ml	2.5 ml	12.5 ml	DM250	0.025%	250 mg/L	1.39 µM
2.5 ml	2.75 ml	5 ml	25 ml	DM500	0.05%	500 mg/L	2.78 mM
5 ml	5.5 ml	10 ml	50 ml	DM1000	0.1%	1000 mg/L	5.55 mM
10 ml	11 ml	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 µM	Glucose (in DM25)

Included topic: ProtocolsRecipesDavisMingioli

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Included topic: ProtocolsRecipesNZY+Broth

M9 Minimal Medium

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-components 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/SOC: Super Optimal Broth

For SOB:

200mL	250mL	1L	Final [ ]	Component
1 g	1.25 g	5 g	0.5%	yeast extract
4 g	5 g	20 g	2.0%	tryptone
0.12 g	0.125 g	0.5 g	10 mM	NaCl
0.037 g*	0.042 g*	0.186 g	2 mM	KCl
0.48 g	0.6 g	2.4 g	20 mM	MgSO4 (anhydrous)

*Don't measure; it's just a pinch.

Adjust to pH 7.5 with 1M NaOH.

Note: 200 ml of SOC makes 25 vials of 8 ml each.

For 1 L: 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 1 L.

Autoclave.

For SOC (=SOB+glucose)

Bulk scale: Make SOB, except add dH₂O to a final volume of 960 ml instead of 1 L. Autoclave. When solution has cooled to 50°C (cool enough to touch with bare hands for a few seconds), add 40 ml of sterile 10% (w/v) glucose. (That's a final concentration of 0.4% glucose or about 20 mM.)

Smaller scale: To achieve a 20 mM concentration of glucose for a known final volume n of SOC, you can calculate the amount of 10% w/v glucose to be added as 0.036n. The amount of SOB to add is then 0.964n. Keep units constant, so if n is in µL, the final amounts will also be in µL and so on.

• Note: If you have a known volume of SOB x and want to know how much 10% w/v glucose to add to make SOC containing 20 mM glucose, simply divide x/0.964 to get your final volume n and subtract x from n to get the volume of 10% w/v glucose to add.

Generally when making stocks of SOC it is advisable to make smaller volume aliquots rather than larger ones. SOC media is extremely rich and can contaminate easily particularly after glucose is added. Additionally, smaller volumes tend to be pulled from it repeatedly with pipettes, which increases the chances of contamination over time.

Note: Some recipes for SOB use 10 mM MgSO₄ and 10 mM MgCl.

Included topic: ProtocolsRecipesSOB

Warning: Can't find topic Lab.ProtocolsRecipesSOC

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

S2

Used for Acinetobacter

Recipe for 900mL

(Autoclave in three separate bottles - 300mL each)

Bottle 1 (Erlenmeyer flask: 300mL)

300mL	Component
1.35g	KH2PO4 (monobasic)
22.95g	Na2HPO4 7H2O

Bottle 2 (Erlenmeyer flask: 300mL)

300mL	Component
0.09g	MgSO4

Bottle 3 (1L bottle: 300mL)

300mL	Component
1.8g	NH4Cl
81ul	1M CaCl2

After autoclaving, let these reach room temperature and transfer all into one 1L bottle (Bottle 3), then, finally add the following sterile components:

0.45ml	0.1% FeSO4 7H2O
4.5 ml	90% Lactic Acid

The functional constraints are that Calcium Chloride cannot be autoclaved in the presence of sulfates, or it will precipitate as calcium sulfate. Ferrous sulfate when autoclaved will decompose to ferric sulfate, ferric oxide and sulfur dioxide. This means that either calcium chloride OR magnesium sulfate may be added before autoclaving, but not both.

Final composition
190.2 mM	Sodium (Na+)
11.0 mM	Potassium (K+)
37.8 mM	Ammonium (NH4+)
0.09 mM	Calcium (Ca2+)
0.83 mM	Magnesium (Mg2+)
0.033 mM	Iron II (Fe2+)
38.0 mM	Chloride (Cl-)
0.87 mM	Sulfate (SO42-)
106.1 mM	Phosphate (PO43-)
50.0 mM	Lactic Acid

-- Main.GabrielSuarez - 05 Sep 2013

Included topic:ProtocolsRecipesS2

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

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Included topic: ProtocolsRecipesLuria-Broth

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

Combine in a 1 L flask:

500 mL	Component
5 g	Tryptone
500 mg	Yeast Extract
2.5 g	NaCl
8 g	Agar
500 µL	5% Antifoam

Add dH₂O to 433.33 mL.

Combine in a 125 ml flask:

500 mL	Component
5 g	Sugar (arabinose = TA, maltose = TM, lactose = TL)

Add dH₂O to 66.67 mL.

Cover each flask with foil, and autoclave sugar and media separately. Total combined volume will be 500 mL.

Combine the autoclaved sugar and media solutions and add:

500 mL	Component
500 µL	5% (w/v) Triphenyl Tetrazolium chloride (TTC), filter sterilized, stored at 4°C

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.

500 mL	1.0L	Component
2.65 g	5.3 g	Potassium Phosphate (dibasic) K2HPO4
1 g	2 g	Potassium Phosphate (monobasic) KH2PO4
0.5 g	1 g	Ammonium Sulfate (NH4)2 SO4
0.25 g	0.5 g	Sodium Citrate (trisodium, dihydrate) Na3C6H5O7 (H2O)2
166.7 mL	333 mL	dH2O

Separately, prepare the agar:

500 mL	1.0 L	Component
8 g	16 g	Agar
500 µL	1 mL	Antifoam (5%)
166.7 mL	333 mL	dH2O

Next, prepare the sugar solution. The final concentration is 0.4% w/v:

500 mL	1.0 L	Component
2 g	4 g	Glucose, arabinose, or other sugar
166.7 mL	333 mL	dH2O

Autoclave the 3 parts separately for a 40 minute sterilization time.

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:

500 mL	1.0 L	Component
500 µL	1.0 ml	10% MgSO4 (separately autoclaved stock)
500 µL	1.0 ml	0.2% Thiamine (vitamin B1) (filter sterilized stock refrigerated at 4ºC)

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

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Included topic: ProtocolsRecipesM9

M9 Minimal Media Plates

As with DM and MG media, make sure to autoclave the agar and phosphate separately.

For 1 liter of media:

1L	Component
6 g	Sodium phosphate, Na2HPO4 (anhydrous)
3 g	Potassium phosphate, KH2PO4
0.5 g	Sodium chloride, NaCl
1 g	Ammonium chloride, NH4Cl

Add dH₂O to 500 mL

1L	Component
16 g	Agar
1 ml	Antifoam

Add dH₂O to 500 mL.

Autoclave media. Once the bottles are cooled to 55°C, mix the agar and M9 components.

Next, 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: 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 G•C→T•A 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