Revision 482025-06-12 - AlexaMorton

META TOPICPARENT	name="ProtocolsMediaRecipes"

Media Recipes

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When making media, make sure the autoclave bin you're using contains water (DI or tap) at least half an inch deep to help prevent excess water loss from the media itself in the autoclave.

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.

Add dH₂O to desired volume in an appropriate flask. Combine the following, making sure the components completely dissolve before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl

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. For 1L bottles, add 500 ml of LB to each. Each liter of LB makes 2 bottles. Autoclave.

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.

0.5L	1L	4.5L	5L	Component	MW
2.67 g	5.34 g	24.03 g	26.7 g	Potassium Phosphate (dibasic) K₂HPO₄	MW 174.18
1 g	2 g	9 g	10 g	Potassium Phosphate (monobasic) KH₂PO₄	MW 136.07
0.5 g	1 g	4.5 g	5 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.25 g	0.5 g	2.25 g	2.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to final volume and autoclave.

Note: 4.5 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 Heptahydrate MgSO₄•7H₂O (separately autoclaved stock)
0.5 ml	550 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)
0.406 mM	Magnesium (Mg²⁺)
8.41 mM	Sulfate (SO₄^2-)
45.3 mM	Phosphate (PO₄^3-)
1.70 mM	Citrate
139 µM	Glucose (in DM25)

Note: Recipe was corrected on 2026-04-22 to use MgSO₄•7H₂O.

Included topic: ProtocolsRecipesDavisMingioli

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

Included topic: ProtocolsRecipesM9Minimal

ABMS: Acinetobacter Minimal Succinate

Courtesy of the Averhoff lab, with modifications for available reagents.

To make standard ABMS, combine the following pre-sterilized ingredients at room temperature:

1L	5L	Component
880 mL	4.4 L	diH₂O
20 mL	100 mL	1M Sodium Succinate
50 mL	250 mL	20× Mineral Solution
50 mL	250 mL	20× Phosphate Buffer

To make ABMS agar:

Per liter of media, add 16 g/L agar and 1 mL/L antifoam to diH₂O, then autoclave in a 2 L flask. When water and agar are cool enough to touch, add component solutions. Optional: warm components at 55°C in a water bath before combining with agar to reduce the temperature difference when combined.

ABMS Component Recipes

SL9 (trace minerals)

1L	Component
800 mL	diH₂O
12.8 g	Nitrilotriacetic acid
2 g	FeSO₄•7H₂O (note: first dissolve in 2M HCl)
104 mg	CoCl₂ (anhydrous)
122 mg	MnCl₂•4H₂O
70 mg	ZnCl₂
36 mg	Na₂MoO₄•2H₂O
13 mg	NiCl₂

Add 1 mL of 10 mL 10× solution:

60 mg	H₃BO₃
20 mg	CuCl₂•2H₂O

To dissolve all components, adjust pH to ~7 or slightly higher using 1 M NaOH while you bring the volume to 1000 mL with diH₂O. Note: this requires a lot of NaOH, so leave room when bringing up to volume.

Mineral Solution (20×)

500 mL	Component
10 g	NH₄Cl
5.8 g	MgSO₄•7H₂O
1 g	KNO₃
0.67 g	CaCl₂•2H₂O †
20 mg	(NH₄)₆Mo₇O₂₄•4H₂O
10 mL	SL 9

† Substitution: You may use 465.8 µL of a 1 M CaCl₂ per 500 ml of stock solution for this component. The final concentration of Ca²⁺ in the 20× stock is 9.12 mM.

Bring to 500mL in a graduated cylinder with deionized water.

Phosphate Buffer (20×)

500mL	Components
68 g	KH₂PO₄
132.5 g	Na₂HPO₄•H₂O

Bring to 500 mL with diH₂O; pH should be ~6.9-7.0 (you may add 400 mL diH₂O first and bring pH up to 6.8 then complete volume to 500mL)

* Source: Averhoff lab

Included topic: ProtocolsRecipesABMS

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

Included topic: ProtocolsRecipesSOB

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

YPD Medium

General media used for culturing Yeast. Adapted from Cold spring harbor

In 1 L bottle:

1L	Component
20g	Peptone
10 g	Yeast Extract

Dissolve above in ~900 mL water.
Bring to 950 mL (final volume will be 1L after autoclaving)

In separate 500mL bottle, prepare 40% Glucose w/v

Autoclave separately. WARNING If glucose added before autoclaving, media will caramelize and be unusable.

Add 50 mL of 40% glucose solution to YPD media.

Included topic:ProtocolsRecipesYPDmedium

Czapek Broth (CB)

Combine in a flask:

500 mL	1 L	Component
1.5 g	3 g	sodium nitrate
0.5 g	1 g	potassium phosphate dibasic
0.25 g	0.5 g	potassium chloride
9 mg	18 mg	ferrous sulfate heptahydrate
2.075 mL	4.15 mL	1 M sterile magnesium sulfate

In a flask, combine sodium nitrate, potassium phosphate dibasic, potassium chloride, and ferrous sulfate heptahydrate in diH₂O
- If making 1 L of media, use 995.85 mL diH₂O
- If making 500 mL of media, use 497.925 mL diH₂O
Once reagents are combined and dissolved, measure pH and adjust until it reaches 7.5
Once pH of 7.5 is achieved, autoclave the solution and allow to cool
Once cooled, add 1 M magnesium sulfate

Source: Throne-Holst et. al 2007

Included topic:ProtocolsRecipesCzapekBroth

Solid Media

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

Add dH₂O to full volume in an appropriately-sized flask. Combine the following, making sure each component fully dissolves before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl
1 ml	6 ml	5% Antifoam*
16 g	48 g	Agar*

ANTIFOAM: Open the bottle under sterile conditions (near a flame) only, and continue agitating after the addition of the 5% antifoam.

AGAR: Measure 8 grams of agar per 500 ml bottle and add to each individual bottle before aliquoting the LB. Autoclave to homogenize.

Each liter of LB Agar makes 2 bottles of 500 ml.

Notes and variations:

Common synonyms are Luria-Broth and Luria-Bertani medium.
This is the "Miller" formulation of LB with 10 g NaCl per liter (1% w/v). Another common formulation has only 5 g NaCl per liter (0.5% w/v)! They are NOT interchangeable.
If you are performing sacB counter-selection, you do not want to include NaCl in your agar. Use the above recipe, but swap out 5-10% w/v (50-100g per liter) sucrose in place of any NaCl. Sucrose can be directly combined with the other components and autoclaved (no need to filter sterilize it separately).
To make "soft" or "top" agar for phage studies, use only 7-8 g agar per liter.

Included topic: ProtocolsRecipesLysogenyBrothAgar

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

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, Na₂HPO_{4 (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: ProtocolsRecipesM9

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:

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.
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

MOB: Mobility Media

This media is useful for enhancing the mobility of ADP1, which moves across the plate by "twitching".

1L	Final [ ]	Component
5 g	0.5%	agarose
5 g	2.0%	tryptone
2.5 g	10 mM	NaCl

After pouring your gel, let it set overnight at room temperature. The next day, spot with 1µl of your overnight-grown culture, leave the plate to dry for 15 minutes at room temperature, wrap in Parafilm, invert, and incubate at 30°C. You should see spreading over the next 24 hours.

Included topic: ProtocolsMobilityMedia

Blood - Heart Infusion Agar

Heart Infusion Agar supplemented with sterile Sheep's Blood. Used for the cultivation of fastidious organisms (Bee gut microbiome species et al)

1L	Component
44 g	Heart Infusion Agar (HIA) (Difco brand)
50mL	Sterile Sheep's Blood

Autoclave water and media (without blood) on a 20min liquid cycle. Transfer autoclaved media to hot water bath at 55 deg Celsius. Allow to equilibrate for 30mins to 1hr. Add 100mL blood (using PipetAid) and stir to mix (a stir bar isn't necessary, but do not shake). Smooth, small circles while the bottle is on a flat surface is sufficient to mix the blood and warm agar. Add necessary antibiotics and supplements (remember the volume is now 1100 mL, not 1 L) and pour into plates.

As with other agar, after autoclaving the HIA you can let it solidify at room temperature. After microwaving to dissolve, it still needs to be cooled before adding blood.

Included topic: ProtocolsRecipesBloodHeartInfusion

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

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Revision 472024-10-16 - MeghnaVergis

META TOPICPARENT	name="ProtocolsMediaRecipes"

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.

Add dH₂O to desired volume in an appropriate flask. Combine the following, making sure the components completely dissolve before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl

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. For 1L bottles, add 500 ml of LB to each. Each liter of LB makes 2 bottles. Autoclave.

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.

0.5L	1L	4.5L	5L	Component	MW
2.67 g	5.34 g	24.03 g	26.7 g	Potassium Phosphate (dibasic) K₂HPO₄	MW 174.18
1 g	2 g	9 g	10 g	Potassium Phosphate (monobasic) KH₂PO₄	MW 136.07
0.5 g	1 g	4.5 g	5 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.25 g	0.5 g	2.25 g	2.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to final volume and autoclave.

Note: 4.5 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 Heptahydrate MgSO₄•7H₂O (separately autoclaved stock)
0.5 ml	550 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)
0.406 mM	Magnesium (Mg²⁺)
8.41 mM	Sulfate (SO₄^2-)
45.3 mM	Phosphate (PO₄^3-)
1.70 mM	Citrate
139 µM	Glucose (in DM25)

Note: Recipe was corrected on 2026-04-22 to use MgSO₄•7H₂O.

Included topic: ProtocolsRecipesDavisMingioli

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

Included topic: ProtocolsRecipesM9Minimal

ABMS: Acinetobacter Minimal Succinate

Courtesy of the Averhoff lab, with modifications for available reagents.

To make standard ABMS, combine the following pre-sterilized ingredients at room temperature:

1L	5L	Component
880 mL	4.4 L	diH₂O
20 mL	100 mL	1M Sodium Succinate
50 mL	250 mL	20× Mineral Solution
50 mL	250 mL	20× Phosphate Buffer

To make ABMS agar:

Per liter of media, add 16 g/L agar and 1 mL/L antifoam to diH₂O, then autoclave in a 2 L flask. When water and agar are cool enough to touch, add component solutions. Optional: warm components at 55°C in a water bath before combining with agar to reduce the temperature difference when combined.

ABMS Component Recipes

SL9 (trace minerals)

1L	Component
800 mL	diH₂O
12.8 g	Nitrilotriacetic acid
2 g	FeSO₄•7H₂O (note: first dissolve in 2M HCl)
104 mg	CoCl₂ (anhydrous)
122 mg	MnCl₂•4H₂O
70 mg	ZnCl₂
36 mg	Na₂MoO₄•2H₂O
13 mg	NiCl₂

Add 1 mL of 10 mL 10× solution:

60 mg	H₃BO₃
20 mg	CuCl₂•2H₂O

To dissolve all components, adjust pH to ~7 or slightly higher using 1 M NaOH while you bring the volume to 1000 mL with diH₂O. Note: this requires a lot of NaOH, so leave room when bringing up to volume.

Mineral Solution (20×)

500 mL	Component
10 g	NH₄Cl
5.8 g	MgSO₄•7H₂O
1 g	KNO₃
0.67 g	CaCl₂•2H₂O †
20 mg	(NH₄)₆Mo₇O₂₄•4H₂O
10 mL	SL 9

† Substitution: You may use 465.8 µL of a 1 M CaCl₂ per 500 ml of stock solution for this component. The final concentration of Ca²⁺ in the 20× stock is 9.12 mM.

Bring to 500mL in a graduated cylinder with deionized water.

Phosphate Buffer (20×)

500mL	Components
68 g	KH₂PO₄
132.5 g	Na₂HPO₄•H₂O

Bring to 500 mL with diH₂O; pH should be ~6.9-7.0 (you may add 400 mL diH₂O first and bring pH up to 6.8 then complete volume to 500mL)

* Source: Averhoff lab

Included topic: ProtocolsRecipesABMS

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

Included topic: ProtocolsRecipesSOB

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

YPD Medium

General media used for culturing Yeast. Adapted from Cold spring harbor

In 1 L bottle:

1L	Component
20g	Peptone
10 g	Yeast Extract

Dissolve above in ~900 mL water.
Bring to 950 mL (final volume will be 1L after autoclaving)

In separate 500mL bottle, prepare 40% Glucose w/v

Autoclave separately. WARNING If glucose added before autoclaving, media will caramelize and be unusable.

Add 50 mL of 40% glucose solution to YPD media.

Included topic:ProtocolsRecipesYPDmedium

Czapek Broth (CB)

Combine in a flask:

500 mL	1 L	Component
1.5 g	3 g	sodium nitrate
0.5 g	1 g	potassium phosphate dibasic
0.25 g	0.5 g	potassium chloride
9 mg	18 mg	ferrous sulfate heptahydrate
2.075 mL	4.15 mL	1 M sterile magnesium sulfate

In a flask, combine sodium nitrate, potassium phosphate dibasic, potassium chloride, and ferrous sulfate heptahydrate in diH₂O
- If making 1 L of media, use 995.85 mL diH₂O
- If making 500 mL of media, use 497.925 mL diH₂O
Once reagents are combined and dissolved, measure pH and adjust until it reaches 7.5
Once pH of 7.5 is achieved, autoclave the solution and allow to cool
Once cooled, add 1 M magnesium sulfate

Source: Throne-Holst et. al 2007

Included topic:ProtocolsRecipesCzapekBroth

Solid Media

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

Add dH₂O to full volume in an appropriately-sized flask. Combine the following, making sure each component fully dissolves before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl
1 ml	6 ml	5% Antifoam*
16 g	48 g	Agar*

ANTIFOAM: Open the bottle under sterile conditions (near a flame) only, and continue agitating after the addition of the 5% antifoam.

AGAR: Measure 8 grams of agar per 500 ml bottle and add to each individual bottle before aliquoting the LB. Autoclave to homogenize.

Each liter of LB Agar makes 2 bottles of 500 ml.

Notes and variations:

Common synonyms are Luria-Broth and Luria-Bertani medium.
This is the "Miller" formulation of LB with 10 g NaCl per liter (1% w/v). Another common formulation has only 5 g NaCl per liter (0.5% w/v)! They are NOT interchangeable.
If you are performing sacB counter-selection, you do not want to include NaCl in your agar. Use the above recipe, but swap out 5-10% w/v (50-100g per liter) sucrose in place of any NaCl. Sucrose can be directly combined with the other components and autoclaved (no need to filter sterilize it separately).
To make "soft" or "top" agar for phage studies, use only 7-8 g agar per liter.

Included topic: ProtocolsRecipesLysogenyBrothAgar

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

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, Na₂HPO_{4 (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: ProtocolsRecipesM9

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:

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.
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

MOB: Mobility Media

This media is useful for enhancing the mobility of ADP1, which moves across the plate by "twitching".

1L	Final [ ]	Component
5 g	0.5%	agarose
5 g	2.0%	tryptone
2.5 g	10 mM	NaCl

After pouring your gel, let it set overnight at room temperature. The next day, spot with 1µl of your overnight-grown culture, leave the plate to dry for 15 minutes at room temperature, wrap in Parafilm, invert, and incubate at 30°C. You should see spreading over the next 24 hours.

Included topic: ProtocolsMobilityMedia

Blood - Heart Infusion Agar

Heart Infusion Agar supplemented with sterile Sheep's Blood. Used for the cultivation of fastidious organisms (Bee gut microbiome species et al)

1L	Component
44 g	Heart Infusion Agar (HIA) (Difco brand)
50mL	Sterile Sheep's Blood

Autoclave water and media (without blood) on a 20min liquid cycle. Transfer autoclaved media to hot water bath at 55 deg Celsius. Allow to equilibrate for 30mins to 1hr. Add 100mL blood (using PipetAid) and stir to mix (a stir bar isn't necessary, but do not shake). Smooth, small circles while the bottle is on a flat surface is sufficient to mix the blood and warm agar. Add necessary antibiotics and supplements (remember the volume is now 1100 mL, not 1 L) and pour into plates.

As with other agar, after autoclaving the HIA you can let it solidify at room temperature. After microwaving to dissolve, it still needs to be cooled before adding blood.

Included topic: ProtocolsRecipesBloodHeartInfusion

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

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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.

Add dH₂O to desired volume in an appropriate flask. Combine the following, making sure the components completely dissolve before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl

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. For 1L bottles, add 500 ml of LB to each. Each liter of LB makes 2 bottles. Autoclave.

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.

0.5L	1L	4.5L	5L	Component	MW
2.67 g	5.34 g	24.03 g	26.7 g	Potassium Phosphate (dibasic) K₂HPO₄	MW 174.18
1 g	2 g	9 g	10 g	Potassium Phosphate (monobasic) KH₂PO₄	MW 136.07
0.5 g	1 g	4.5 g	5 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.25 g	0.5 g	2.25 g	2.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to final volume and autoclave.

Note: 4.5 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 Heptahydrate MgSO₄•7H₂O (separately autoclaved stock)
0.5 ml	550 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)
0.406 mM	Magnesium (Mg²⁺)
8.41 mM	Sulfate (SO₄^2-)
45.3 mM	Phosphate (PO₄^3-)
1.70 mM	Citrate
139 µM	Glucose (in DM25)

Note: Recipe was corrected on 2026-04-22 to use MgSO₄•7H₂O.

Included topic: ProtocolsRecipesDavisMingioli

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

Included topic: ProtocolsRecipesM9Minimal

ABMS: Acinetobacter Minimal Succinate

Courtesy of the Averhoff lab, with modifications for available reagents.

To make standard ABMS, combine the following pre-sterilized ingredients at room temperature:

1L	5L	Component
880 mL	4.4 L	diH₂O
20 mL	100 mL	1M Sodium Succinate
50 mL	250 mL	20× Mineral Solution
50 mL	250 mL	20× Phosphate Buffer

To make ABMS agar:

Per liter of media, add 16 g/L agar and 1 mL/L antifoam to diH₂O, then autoclave in a 2 L flask. When water and agar are cool enough to touch, add component solutions. Optional: warm components at 55°C in a water bath before combining with agar to reduce the temperature difference when combined.

ABMS Component Recipes

SL9 (trace minerals)

1L	Component
800 mL	diH₂O
12.8 g	Nitrilotriacetic acid
2 g	FeSO₄•7H₂O (note: first dissolve in 2M HCl)
104 mg	CoCl₂ (anhydrous)
122 mg	MnCl₂•4H₂O
70 mg	ZnCl₂
36 mg	Na₂MoO₄•2H₂O
13 mg	NiCl₂

Add 1 mL of 10 mL 10× solution:

60 mg	H₃BO₃
20 mg	CuCl₂•2H₂O

To dissolve all components, adjust pH to ~7 or slightly higher using 1 M NaOH while you bring the volume to 1000 mL with diH₂O. Note: this requires a lot of NaOH, so leave room when bringing up to volume.

Mineral Solution (20×)

500 mL	Component
10 g	NH₄Cl
5.8 g	MgSO₄•7H₂O
1 g	KNO₃
0.67 g	CaCl₂•2H₂O †
20 mg	(NH₄)₆Mo₇O₂₄•4H₂O
10 mL	SL 9

† Substitution: You may use 465.8 µL of a 1 M CaCl₂ per 500 ml of stock solution for this component. The final concentration of Ca²⁺ in the 20× stock is 9.12 mM.

Bring to 500mL in a graduated cylinder with deionized water.

Phosphate Buffer (20×)

500mL	Components
68 g	KH₂PO₄
132.5 g	Na₂HPO₄•H₂O

Bring to 500 mL with diH₂O; pH should be ~6.9-7.0 (you may add 400 mL diH₂O first and bring pH up to 6.8 then complete volume to 500mL)

* Source: Averhoff lab

Included topic: ProtocolsRecipesABMS

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

Included topic: ProtocolsRecipesSOB

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

YPD Medium

General media used for culturing Yeast. Adapted from Cold spring harbor

In 1 L bottle:

1L	Component
20g	Peptone
10 g	Yeast Extract

Dissolve above in ~900 mL water.
Bring to 950 mL (final volume will be 1L after autoclaving)

In separate 500mL bottle, prepare 40% Glucose w/v

Autoclave separately. WARNING If glucose added before autoclaving, media will caramelize and be unusable.

Add 50 mL of 40% glucose solution to YPD media.

Included topic:ProtocolsRecipesYPDmedium

Added:

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Czapek Broth (CB)

Combine in a flask:

500 mL	1 L	Component
1.5 g	3 g	sodium nitrate
0.5 g	1 g	potassium phosphate dibasic
0.25 g	0.5 g	potassium chloride
9 mg	18 mg	ferrous sulfate heptahydrate
2.075 mL	4.15 mL	1 M sterile magnesium sulfate

In a flask, combine sodium nitrate, potassium phosphate dibasic, potassium chloride, and ferrous sulfate heptahydrate in diH₂O
- If making 1 L of media, use 995.85 mL diH₂O
- If making 500 mL of media, use 497.925 mL diH₂O
Once reagents are combined and dissolved, measure pH and adjust until it reaches 7.5
Once pH of 7.5 is achieved, autoclave the solution and allow to cool
Once cooled, add 1 M magnesium sulfate

Source: Throne-Holst et. al 2007

Included topic:ProtocolsRecipesCzapekBroth

Solid Media

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

Add dH₂O to full volume in an appropriately-sized flask. Combine the following, making sure each component fully dissolves before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl
1 ml	6 ml	5% Antifoam*
16 g	48 g	Agar*

ANTIFOAM: Open the bottle under sterile conditions (near a flame) only, and continue agitating after the addition of the 5% antifoam.

AGAR: Measure 8 grams of agar per 500 ml bottle and add to each individual bottle before aliquoting the LB. Autoclave to homogenize.

Each liter of LB Agar makes 2 bottles of 500 ml.

Notes and variations:

Common synonyms are Luria-Broth and Luria-Bertani medium.
This is the "Miller" formulation of LB with 10 g NaCl per liter (1% w/v). Another common formulation has only 5 g NaCl per liter (0.5% w/v)! They are NOT interchangeable.
If you are performing sacB counter-selection, you do not want to include NaCl in your agar. Use the above recipe, but swap out 5-10% w/v (50-100g per liter) sucrose in place of any NaCl. Sucrose can be directly combined with the other components and autoclaved (no need to filter sterilize it separately).
To make "soft" or "top" agar for phage studies, use only 7-8 g agar per liter.

Included topic: ProtocolsRecipesLysogenyBrothAgar

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

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, Na₂HPO_{4 (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: ProtocolsRecipesM9

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:

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.
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

MOB: Mobility Media

This media is useful for enhancing the mobility of ADP1, which moves across the plate by "twitching".

1L	Final [ ]	Component
5 g	0.5%	agarose
5 g	2.0%	tryptone
2.5 g	10 mM	NaCl

After pouring your gel, let it set overnight at room temperature. The next day, spot with 1µl of your overnight-grown culture, leave the plate to dry for 15 minutes at room temperature, wrap in Parafilm, invert, and incubate at 30°C. You should see spreading over the next 24 hours.

Included topic: ProtocolsMobilityMedia

Blood - Heart Infusion Agar

Heart Infusion Agar supplemented with sterile Sheep's Blood. Used for the cultivation of fastidious organisms (Bee gut microbiome species et al)

1L	Component
44 g	Heart Infusion Agar (HIA) (Difco brand)
50mL	Sterile Sheep's Blood

Autoclave water and media (without blood) on a 20min liquid cycle. Transfer autoclaved media to hot water bath at 55 deg Celsius. Allow to equilibrate for 30mins to 1hr. Add 100mL blood (using PipetAid) and stir to mix (a stir bar isn't necessary, but do not shake). Smooth, small circles while the bottle is on a flat surface is sufficient to mix the blood and warm agar. Add necessary antibiotics and supplements (remember the volume is now 1100 mL, not 1 L) and pour into plates.

As with other agar, after autoclaving the HIA you can let it solidify at room temperature. After microwaving to dissolve, it still needs to be cooled before adding blood.

Included topic: ProtocolsRecipesBloodHeartInfusion

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

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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.

Add dH₂O to desired volume in an appropriate flask. Combine the following, making sure the components completely dissolve before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl

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. For 1L bottles, add 500 ml of LB to each. Each liter of LB makes 2 bottles. Autoclave.

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.

0.5L	1L	4.5L	5L	Component	MW
2.67 g	5.34 g	24.03 g	26.7 g	Potassium Phosphate (dibasic) K₂HPO₄	MW 174.18
1 g	2 g	9 g	10 g	Potassium Phosphate (monobasic) KH₂PO₄	MW 136.07
0.5 g	1 g	4.5 g	5 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.25 g	0.5 g	2.25 g	2.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to final volume and autoclave.

Note: 4.5 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 Heptahydrate MgSO₄•7H₂O (separately autoclaved stock)
0.5 ml	550 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)
0.406 mM	Magnesium (Mg²⁺)
8.41 mM	Sulfate (SO₄^2-)
45.3 mM	Phosphate (PO₄^3-)
1.70 mM	Citrate
139 µM	Glucose (in DM25)

Note: Recipe was corrected on 2026-04-22 to use MgSO₄•7H₂O.

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

Included topic: ProtocolsRecipesM9Minimal

ABMS: Acinetobacter Minimal Succinate

Courtesy of the Averhoff lab, with modifications for available reagents.

To make standard ABMS, combine the following pre-sterilized ingredients at room temperature:

1L	5L	Component
880 mL	4.4 L	diH₂O
20 mL	100 mL	1M Sodium Succinate
50 mL	250 mL	20× Mineral Solution
50 mL	250 mL	20× Phosphate Buffer

To make ABMS agar:

Per liter of media, add 16 g/L agar and 1 mL/L antifoam to diH₂O, then autoclave in a 2 L flask. When water and agar are cool enough to touch, add component solutions. Optional: warm components at 55°C in a water bath before combining with agar to reduce the temperature difference when combined.

ABMS Component Recipes

SL9 (trace minerals)

1L	Component
800 mL	diH₂O
12.8 g	Nitrilotriacetic acid
2 g	FeSO₄•7H₂O (note: first dissolve in 2M HCl)
104 mg	CoCl₂ (anhydrous)
122 mg	MnCl₂•4H₂O
70 mg	ZnCl₂
36 mg	Na₂MoO₄•2H₂O
13 mg	NiCl₂

Add 1 mL of 10 mL 10× solution:

60 mg	H₃BO₃
20 mg	CuCl₂•2H₂O

To dissolve all components, adjust pH to ~7 or slightly higher using 1 M NaOH while you bring the volume to 1000 mL with diH₂O. Note: this requires a lot of NaOH, so leave room when bringing up to volume.

Mineral Solution (20×)

500 mL	Component
10 g	NH₄Cl
5.8 g	MgSO₄•7H₂O
1 g	KNO₃
0.67 g	CaCl₂•2H₂O †
20 mg	(NH₄)₆Mo₇O₂₄•4H₂O
10 mL	SL 9

† Substitution: You may use 465.8 µL of a 1 M CaCl₂ per 500 ml of stock solution for this component. The final concentration of Ca²⁺ in the 20× stock is 9.12 mM.

Bring to 500mL in a graduated cylinder with deionized water.

Phosphate Buffer (20×)

500mL	Components
68 g	KH₂PO₄
132.5 g	Na₂HPO₄•H₂O

Bring to 500 mL with diH₂O; pH should be ~6.9-7.0 (you may add 400 mL diH₂O first and bring pH up to 6.8 then complete volume to 500mL)

* Source: Averhoff lab

Included topic: ProtocolsRecipesABMS

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

Included topic: ProtocolsRecipesSOB

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

YPD Medium

General media used for culturing Yeast. Adapted from Cold spring harbor

In 1 L bottle:

1L	Component
20g	Peptone
10 g	Yeast Extract

Dissolve above in ~900 mL water.
Bring to 950 mL (final volume will be 1L after autoclaving)

In separate 500mL bottle, prepare 40% Glucose w/v

Autoclave separately. WARNING If glucose added before autoclaving, media will caramelize and be unusable.

Add 50 mL of 40% glucose solution to YPD media.

Included topic:ProtocolsRecipesYPDmedium

Solid Media

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

Add dH₂O to full volume in an appropriately-sized flask. Combine the following, making sure each component fully dissolves before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl
1 ml	6 ml	5% Antifoam*
16 g	48 g	Agar*

ANTIFOAM: Open the bottle under sterile conditions (near a flame) only, and continue agitating after the addition of the 5% antifoam.

AGAR: Measure 8 grams of agar per 500 ml bottle and add to each individual bottle before aliquoting the LB. Autoclave to homogenize.

Each liter of LB Agar makes 2 bottles of 500 ml.

Notes and variations:

Common synonyms are Luria-Broth and Luria-Bertani medium.
This is the "Miller" formulation of LB with 10 g NaCl per liter (1% w/v). Another common formulation has only 5 g NaCl per liter (0.5% w/v)! They are NOT interchangeable.
If you are performing sacB counter-selection, you do not want to include NaCl in your agar. Use the above recipe, but swap out 5-10% w/v (50-100g per liter) sucrose in place of any NaCl. Sucrose can be directly combined with the other components and autoclaved (no need to filter sterilize it separately).
To make "soft" or "top" agar for phage studies, use only 7-8 g agar per liter.

Included topic: ProtocolsRecipesLysogenyBrothAgar

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

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, Na₂HPO_{4 (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: ProtocolsRecipesM9

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:

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.
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

MOB: Mobility Media

This media is useful for enhancing the mobility of ADP1, which moves across the plate by "twitching".

1L	Final [ ]	Component
5 g	0.5%	agarose
5 g	2.0%	tryptone
2.5 g	10 mM	NaCl

After pouring your gel, let it set overnight at room temperature. The next day, spot with 1µl of your overnight-grown culture, leave the plate to dry for 15 minutes at room temperature, wrap in Parafilm, invert, and incubate at 30°C. You should see spreading over the next 24 hours.

Included topic: ProtocolsMobilityMedia

Blood - Heart Infusion Agar

Heart Infusion Agar supplemented with sterile Sheep's Blood. Used for the cultivation of fastidious organisms (Bee gut microbiome species et al)

1L	Component
44 g	Heart Infusion Agar (HIA) (Difco brand)
50mL	Sterile Sheep's Blood

Autoclave water and media (without blood) on a 20min liquid cycle. Transfer autoclaved media to hot water bath at 55 deg Celsius. Allow to equilibrate for 30mins to 1hr. Add 100mL blood (using PipetAid) and stir to mix (a stir bar isn't necessary, but do not shake). Smooth, small circles while the bottle is on a flat surface is sufficient to mix the blood and warm agar. Add necessary antibiotics and supplements (remember the volume is now 1100 mL, not 1 L) and pour into plates.

As with other agar, after autoclaving the HIA you can let it solidify at room temperature. After microwaving to dissolve, it still needs to be cooled before adding blood.

Included topic: ProtocolsRecipesBloodHeartInfusion

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

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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.

Add dH₂O to desired volume in an appropriate flask. Combine the following, making sure the components completely dissolve before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl

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. For 1L bottles, add 500 ml of LB to each. Each liter of LB makes 2 bottles. Autoclave.

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.

0.5L	1L	4.5L	5L	Component	MW
2.67 g	5.34 g	24.03 g	26.7 g	Potassium Phosphate (dibasic) K₂HPO₄	MW 174.18
1 g	2 g	9 g	10 g	Potassium Phosphate (monobasic) KH₂PO₄	MW 136.07
0.5 g	1 g	4.5 g	5 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.25 g	0.5 g	2.25 g	2.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to final volume and autoclave.

Note: 4.5 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 Heptahydrate MgSO₄•7H₂O (separately autoclaved stock)
0.5 ml	550 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)
0.406 mM	Magnesium (Mg²⁺)
8.41 mM	Sulfate (SO₄^2-)
45.3 mM	Phosphate (PO₄^3-)
1.70 mM	Citrate
139 µM	Glucose (in DM25)

Note: Recipe was corrected on 2026-04-22 to use MgSO₄•7H₂O.

Included topic: ProtocolsRecipesDavisMingioli

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

Included topic: ProtocolsRecipesM9Minimal

ABMS: Acinetobacter Minimal Succinate

Courtesy of the Averhoff lab, with modifications for available reagents.

To make standard ABMS, combine the following pre-sterilized ingredients at room temperature:

1L	5L	Component
880 mL	4.4 L	diH₂O
20 mL	100 mL	1M Sodium Succinate
50 mL	250 mL	20× Mineral Solution
50 mL	250 mL	20× Phosphate Buffer

To make ABMS agar:

Per liter of media, add 16 g/L agar and 1 mL/L antifoam to diH₂O, then autoclave in a 2 L flask. When water and agar are cool enough to touch, add component solutions. Optional: warm components at 55°C in a water bath before combining with agar to reduce the temperature difference when combined.

ABMS Component Recipes

SL9 (trace minerals)

1L	Component
800 mL	diH₂O
12.8 g	Nitrilotriacetic acid
2 g	FeSO₄•7H₂O (note: first dissolve in 2M HCl)
104 mg	CoCl₂ (anhydrous)
122 mg	MnCl₂•4H₂O
70 mg	ZnCl₂
36 mg	Na₂MoO₄•2H₂O
13 mg	NiCl₂

Add 1 mL of 10 mL 10× solution:

60 mg	H₃BO₃
20 mg	CuCl₂•2H₂O

To dissolve all components, adjust pH to ~7 or slightly higher using 1 M NaOH while you bring the volume to 1000 mL with diH₂O. Note: this requires a lot of NaOH, so leave room when bringing up to volume.

Mineral Solution (20×)

500 mL	Component
10 g	NH₄Cl
5.8 g	MgSO₄•7H₂O
1 g	KNO₃
0.67 g	CaCl₂•2H₂O †
20 mg	(NH₄)₆Mo₇O₂₄•4H₂O
10 mL	SL 9

† Substitution: You may use 465.8 µL of a 1 M CaCl₂ per 500 ml of stock solution for this component. The final concentration of Ca²⁺ in the 20× stock is 9.12 mM.

Bring to 500mL in a graduated cylinder with deionized water.

Phosphate Buffer (20×)

500mL	Components
68 g	KH₂PO₄
132.5 g	Na₂HPO₄•H₂O

Bring to 500 mL with diH₂O; pH should be ~6.9-7.0 (you may add 400 mL diH₂O first and bring pH up to 6.8 then complete volume to 500mL)

* Source: Averhoff lab

Included topic: ProtocolsRecipesABMS

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

Included topic: ProtocolsRecipesSOB

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

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YPD Medium

General media used for culturing Yeast. Adapted from Cold spring harbor

In 1 L bottle:

1L	Component
20g	Peptone
10 g	Yeast Extract

Dissolve above in ~900 mL water.
Bring to 950 mL (final volume will be 1L after autoclaving)

In separate 500mL bottle, prepare 40% Glucose w/v

Autoclave separately. WARNING If glucose added before autoclaving, media will caramelize and be unusable.

Add 50 mL of 40% glucose solution to YPD media.

Included topic:ProtocolsRecipesYPDmedium

Solid Media

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

Add dH₂O to full volume in an appropriately-sized flask. Combine the following, making sure each component fully dissolves before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl
1 ml	6 ml	5% Antifoam*
16 g	48 g	Agar*

ANTIFOAM: Open the bottle under sterile conditions (near a flame) only, and continue agitating after the addition of the 5% antifoam.

AGAR: Measure 8 grams of agar per 500 ml bottle and add to each individual bottle before aliquoting the LB. Autoclave to homogenize.

Each liter of LB Agar makes 2 bottles of 500 ml.

Notes and variations:

Common synonyms are Luria-Broth and Luria-Bertani medium.
This is the "Miller" formulation of LB with 10 g NaCl per liter (1% w/v). Another common formulation has only 5 g NaCl per liter (0.5% w/v)! They are NOT interchangeable.
If you are performing sacB counter-selection, you do not want to include NaCl in your agar. Use the above recipe, but swap out 5-10% w/v (50-100g per liter) sucrose in place of any NaCl. Sucrose can be directly combined with the other components and autoclaved (no need to filter sterilize it separately).
To make "soft" or "top" agar for phage studies, use only 7-8 g agar per liter.

Included topic: ProtocolsRecipesLysogenyBrothAgar

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

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, Na₂HPO_{4 (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: ProtocolsRecipesM9

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:

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.
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

MOB: Mobility Media

This media is useful for enhancing the mobility of ADP1, which moves across the plate by "twitching".

1L	Final [ ]	Component
5 g	0.5%	agarose
5 g	2.0%	tryptone
2.5 g	10 mM	NaCl

After pouring your gel, let it set overnight at room temperature. The next day, spot with 1µl of your overnight-grown culture, leave the plate to dry for 15 minutes at room temperature, wrap in Parafilm, invert, and incubate at 30°C. You should see spreading over the next 24 hours.

Included topic: ProtocolsMobilityMedia

Blood - Heart Infusion Agar

Heart Infusion Agar supplemented with sterile Sheep's Blood. Used for the cultivation of fastidious organisms (Bee gut microbiome species et al)

1L	Component
44 g	Heart Infusion Agar (HIA) (Difco brand)
50mL	Sterile Sheep's Blood

Autoclave water and media (without blood) on a 20min liquid cycle. Transfer autoclaved media to hot water bath at 55 deg Celsius. Allow to equilibrate for 30mins to 1hr. Add 100mL blood (using PipetAid) and stir to mix (a stir bar isn't necessary, but do not shake). Smooth, small circles while the bottle is on a flat surface is sufficient to mix the blood and warm agar. Add necessary antibiotics and supplements (remember the volume is now 1100 mL, not 1 L) and pour into plates.

As with other agar, after autoclaving the HIA you can let it solidify at room temperature. After microwaving to dissolve, it still needs to be cooled before adding blood.

Included topic: ProtocolsRecipesBloodHeartInfusion

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

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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.

Add dH₂O to desired volume in an appropriate flask. Combine the following, making sure the components completely dissolve before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl

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. For 1L bottles, add 500 ml of LB to each. Each liter of LB makes 2 bottles. Autoclave.

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.

0.5L	1L	4.5L	5L	Component	MW
2.67 g	5.34 g	24.03 g	26.7 g	Potassium Phosphate (dibasic) K₂HPO₄	MW 174.18
1 g	2 g	9 g	10 g	Potassium Phosphate (monobasic) KH₂PO₄	MW 136.07
0.5 g	1 g	4.5 g	5 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.25 g	0.5 g	2.25 g	2.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to final volume and autoclave.

Note: 4.5 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 Heptahydrate MgSO₄•7H₂O (separately autoclaved stock)
0.5 ml	550 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)
0.406 mM	Magnesium (Mg²⁺)
8.41 mM	Sulfate (SO₄^2-)
45.3 mM	Phosphate (PO₄^3-)
1.70 mM	Citrate
139 µM	Glucose (in DM25)

Note: Recipe was corrected on 2026-04-22 to use MgSO₄•7H₂O.

Included topic: ProtocolsRecipesDavisMingioli

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

Included topic: ProtocolsRecipesM9Minimal

ABMS: Acinetobacter Minimal Succinate

Courtesy of the Averhoff lab, with modifications for available reagents.

To make standard ABMS, combine the following pre-sterilized ingredients at room temperature:

1L	5L	Component
880 mL	4.4 L	diH₂O
20 mL	100 mL	1M Sodium Succinate
50 mL	250 mL	20× Mineral Solution
50 mL	250 mL	20× Phosphate Buffer

To make ABMS agar:

Per liter of media, add 16 g/L agar and 1 mL/L antifoam to diH₂O, then autoclave in a 2 L flask. When water and agar are cool enough to touch, add component solutions. Optional: warm components at 55°C in a water bath before combining with agar to reduce the temperature difference when combined.

ABMS Component Recipes

SL9 (trace minerals)

1L	Component
800 mL	diH₂O
12.8 g	Nitrilotriacetic acid
2 g	FeSO₄•7H₂O (note: first dissolve in 2M HCl)
104 mg	CoCl₂ (anhydrous)
122 mg	MnCl₂•4H₂O
70 mg	ZnCl₂
36 mg	Na₂MoO₄•2H₂O
13 mg	NiCl₂

Add 1 mL of 10 mL 10× solution:

60 mg	H₃BO₃
20 mg	CuCl₂•2H₂O

To dissolve all components, adjust pH to ~7 or slightly higher using 1 M NaOH while you bring the volume to 1000 mL with diH₂O. Note: this requires a lot of NaOH, so leave room when bringing up to volume.

Mineral Solution (20×)

500 mL	Component
10 g	NH₄Cl
5.8 g	MgSO₄•7H₂O
1 g	KNO₃
0.67 g	CaCl₂•2H₂O †
20 mg	(NH₄)₆Mo₇O₂₄•4H₂O
10 mL	SL 9

† Substitution: You may use 465.8 µL of a 1 M CaCl₂ per 500 ml of stock solution for this component. The final concentration of Ca²⁺ in the 20× stock is 9.12 mM.

Bring to 500mL in a graduated cylinder with deionized water.

Phosphate Buffer (20×)

500mL	Components
68 g	KH₂PO₄
132.5 g	Na₂HPO₄•H₂O

Bring to 500 mL with diH₂O; pH should be ~6.9-7.0 (you may add 400 mL diH₂O first and bring pH up to 6.8 then complete volume to 500mL)

* Source: Averhoff lab

Included topic: ProtocolsRecipesABMS

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

Included topic: ProtocolsRecipesSOB

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

Add dH₂O to full volume in an appropriately-sized flask. Combine the following, making sure each component fully dissolves before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl
1 ml	6 ml	5% Antifoam*
16 g	48 g	Agar*

ANTIFOAM: Open the bottle under sterile conditions (near a flame) only, and continue agitating after the addition of the 5% antifoam.

AGAR: Measure 8 grams of agar per 500 ml bottle and add to each individual bottle before aliquoting the LB. Autoclave to homogenize.

Each liter of LB Agar makes 2 bottles of 500 ml.

Notes and variations:

Common synonyms are Luria-Broth and Luria-Bertani medium.
This is the "Miller" formulation of LB with 10 g NaCl per liter (1% w/v). Another common formulation has only 5 g NaCl per liter (0.5% w/v)! They are NOT interchangeable.
If you are performing sacB counter-selection, you do not want to include NaCl in your agar. Use the above recipe, but swap out 5-10% w/v (50-100g per liter) sucrose in place of any NaCl. Sucrose can be directly combined with the other components and autoclaved (no need to filter sterilize it separately).
To make "soft" or "top" agar for phage studies, use only 7-8 g agar per liter.

Included topic: ProtocolsRecipesLysogenyBrothAgar

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

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, Na₂HPO_{4 (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: ProtocolsRecipesM9

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:

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.
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

MOB: Mobility Media

This media is useful for enhancing the mobility of ADP1, which moves across the plate by "twitching".

1L	Final [ ]	Component
5 g	0.5%	agarose
5 g	2.0%	tryptone
2.5 g	10 mM	NaCl

After pouring your gel, let it set overnight at room temperature. The next day, spot with 1µl of your overnight-grown culture, leave the plate to dry for 15 minutes at room temperature, wrap in Parafilm, invert, and incubate at 30°C. You should see spreading over the next 24 hours.

Included topic: ProtocolsMobilityMedia

Blood - Heart Infusion Agar

Heart Infusion Agar supplemented with sterile Sheep's Blood. Used for the cultivation of fastidious organisms (Bee gut microbiome species et al)

1L	Component
44 g	Heart Infusion Agar (HIA) (Difco brand)
50mL	Sterile Sheep's Blood

Autoclave water and media (without blood) on a 20min liquid cycle. Transfer autoclaved media to hot water bath at 55 deg Celsius. Allow to equilibrate for 30mins to 1hr. Add 100mL blood (using PipetAid) and stir to mix (a stir bar isn't necessary, but do not shake). Smooth, small circles while the bottle is on a flat surface is sufficient to mix the blood and warm agar. Add necessary antibiotics and supplements (remember the volume is now 1100 mL, not 1 L) and pour into plates.

As with other agar, after autoclaving the HIA you can let it solidify at room temperature. After microwaving to dissolve, it still needs to be cooled before adding blood.

Included topic: ProtocolsRecipesBloodHeartInfusion

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

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Revision 422018-12-10 - JeffreyBarrick

META TOPICPARENT	name="ProtocolsMediaRecipes"

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.

Add dH₂O to desired volume in an appropriate flask. Combine the following, making sure the components completely dissolve before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl

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. For 1L bottles, add 500 ml of LB to each. Each liter of LB makes 2 bottles. Autoclave.

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.

0.5L	1L	4.5L	5L	Component	MW
2.67 g	5.34 g	24.03 g	26.7 g	Potassium Phosphate (dibasic) K₂HPO₄	MW 174.18
1 g	2 g	9 g	10 g	Potassium Phosphate (monobasic) KH₂PO₄	MW 136.07
0.5 g	1 g	4.5 g	5 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.25 g	0.5 g	2.25 g	2.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to final volume and autoclave.

Note: 4.5 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 Heptahydrate MgSO₄•7H₂O (separately autoclaved stock)
0.5 ml	550 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)
0.406 mM	Magnesium (Mg²⁺)
8.41 mM	Sulfate (SO₄^2-)
45.3 mM	Phosphate (PO₄^3-)
1.70 mM	Citrate
139 µM	Glucose (in DM25)

Note: Recipe was corrected on 2026-04-22 to use MgSO₄•7H₂O.

Included topic: ProtocolsRecipesDavisMingioli

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

Included topic: ProtocolsRecipesM9Minimal

ABMS: Acinetobacter Minimal Succinate

Courtesy of the Averhoff lab, with modifications for available reagents.

To make standard ABMS, combine the following pre-sterilized ingredients at room temperature:

1L	5L	Component
880 mL	4.4 L	diH₂O
20 mL	100 mL	1M Sodium Succinate
50 mL	250 mL	20× Mineral Solution
50 mL	250 mL	20× Phosphate Buffer

To make ABMS agar:

Per liter of media, add 16 g/L agar and 1 mL/L antifoam to diH₂O, then autoclave in a 2 L flask. When water and agar are cool enough to touch, add component solutions. Optional: warm components at 55°C in a water bath before combining with agar to reduce the temperature difference when combined.

ABMS Component Recipes

SL9 (trace minerals)

1L	Component
800 mL	diH₂O
12.8 g	Nitrilotriacetic acid
2 g	FeSO₄•7H₂O (note: first dissolve in 2M HCl)
104 mg	CoCl₂ (anhydrous)
122 mg	MnCl₂•4H₂O
70 mg	ZnCl₂
36 mg	Na₂MoO₄•2H₂O
13 mg	NiCl₂

Add 1 mL of 10 mL 10× solution:

60 mg	H₃BO₃
20 mg	CuCl₂•2H₂O

To dissolve all components, adjust pH to ~7 or slightly higher using 1 M NaOH while you bring the volume to 1000 mL with diH₂O. Note: this requires a lot of NaOH, so leave room when bringing up to volume.

Mineral Solution (20×)

500 mL	Component
10 g	NH₄Cl
5.8 g	MgSO₄•7H₂O
1 g	KNO₃
0.67 g	CaCl₂•2H₂O †
20 mg	(NH₄)₆Mo₇O₂₄•4H₂O
10 mL	SL 9

† Substitution: You may use 465.8 µL of a 1 M CaCl₂ per 500 ml of stock solution for this component. The final concentration of Ca²⁺ in the 20× stock is 9.12 mM.

Bring to 500mL in a graduated cylinder with deionized water.

Phosphate Buffer (20×)

500mL	Components
68 g	KH₂PO₄
132.5 g	Na₂HPO₄•H₂O

Bring to 500 mL with diH₂O; pH should be ~6.9-7.0 (you may add 400 mL diH₂O first and bring pH up to 6.8 then complete volume to 500mL)

* Source: Averhoff lab

Included topic: ProtocolsRecipesABMS

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

Included topic: ProtocolsRecipesSOB

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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

Add dH₂O to full volume in an appropriately-sized flask. Combine the following, making sure each component fully dissolves before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl
1 ml	6 ml	5% Antifoam*
16 g	48 g	Agar*

ANTIFOAM: Open the bottle under sterile conditions (near a flame) only, and continue agitating after the addition of the 5% antifoam.

AGAR: Measure 8 grams of agar per 500 ml bottle and add to each individual bottle before aliquoting the LB. Autoclave to homogenize.

Each liter of LB Agar makes 2 bottles of 500 ml.

Notes and variations:

Common synonyms are Luria-Broth and Luria-Bertani medium.
This is the "Miller" formulation of LB with 10 g NaCl per liter (1% w/v). Another common formulation has only 5 g NaCl per liter (0.5% w/v)! They are NOT interchangeable.
If you are performing sacB counter-selection, you do not want to include NaCl in your agar. Use the above recipe, but swap out 5-10% w/v (50-100g per liter) sucrose in place of any NaCl. Sucrose can be directly combined with the other components and autoclaved (no need to filter sterilize it separately).
To make "soft" or "top" agar for phage studies, use only 7-8 g agar per liter.

Included topic: ProtocolsRecipesLysogenyBrothAgar

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

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, Na₂HPO_{4 (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: ProtocolsRecipesM9

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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:

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.
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

MOB: Mobility Media

This media is useful for enhancing the mobility of ADP1, which moves across the plate by "twitching".

1L	Final [ ]	Component
5 g	0.5%	agarose
5 g	2.0%	tryptone
2.5 g	10 mM	NaCl

After pouring your gel, let it set overnight at room temperature. The next day, spot with 1µl of your overnight-grown culture, leave the plate to dry for 15 minutes at room temperature, wrap in Parafilm, invert, and incubate at 30°C. You should see spreading over the next 24 hours.

Included topic: ProtocolsMobilityMedia

Blood - Heart Infusion Agar

Heart Infusion Agar supplemented with sterile Sheep's Blood. Used for the cultivation of fastidious organisms (Bee gut microbiome species et al)

1L	Component
44 g	Heart Infusion Agar (HIA) (Difco brand)
50mL	Sterile Sheep's Blood

Autoclave water and media (without blood) on a 20min liquid cycle. Transfer autoclaved media to hot water bath at 55 deg Celsius. Allow to equilibrate for 30mins to 1hr. Add 100mL blood (using PipetAid) and stir to mix (a stir bar isn't necessary, but do not shake). Smooth, small circles while the bottle is on a flat surface is sufficient to mix the blood and warm agar. Add necessary antibiotics and supplements (remember the volume is now 1100 mL, not 1 L) and pour into plates.

As with other agar, after autoclaving the HIA you can let it solidify at room temperature. After microwaving to dissolve, it still needs to be cooled before adding blood.

Included topic: ProtocolsRecipesBloodHeartInfusion

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

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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.

Add dH₂O to desired volume in an appropriate flask. Combine the following, making sure the components completely dissolve before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl

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. For 1L bottles, add 500 ml of LB to each. Each liter of LB makes 2 bottles. Autoclave.

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.

0.5L	1L	4.5L	5L	Component	MW
2.67 g	5.34 g	24.03 g	26.7 g	Potassium Phosphate (dibasic) K₂HPO₄	MW 174.18
1 g	2 g	9 g	10 g	Potassium Phosphate (monobasic) KH₂PO₄	MW 136.07
0.5 g	1 g	4.5 g	5 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.25 g	0.5 g	2.25 g	2.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to final volume and autoclave.

Note: 4.5 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 Heptahydrate MgSO₄•7H₂O (separately autoclaved stock)
0.5 ml	550 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)
0.406 mM	Magnesium (Mg²⁺)
8.41 mM	Sulfate (SO₄^2-)
45.3 mM	Phosphate (PO₄^3-)
1.70 mM	Citrate
139 µM	Glucose (in DM25)

Note: Recipe was corrected on 2026-04-22 to use MgSO₄•7H₂O.

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

Included topic: ProtocolsRecipesM9Minimal

ABMS: Acinetobacter Minimal Succinate

Courtesy of the Averhoff lab, with modifications for available reagents.

To make standard ABMS, combine the following pre-sterilized ingredients at room temperature:

1L	5L	Component
880 mL	4.4 L	diH₂O
20 mL	100 mL	1M Sodium Succinate
50 mL	250 mL	20× Mineral Solution
50 mL	250 mL	20× Phosphate Buffer

To make ABMS agar:

Per liter of media, add 16 g/L agar and 1 mL/L antifoam to diH₂O, then autoclave in a 2 L flask. When water and agar are cool enough to touch, add component solutions. Optional: warm components at 55°C in a water bath before combining with agar to reduce the temperature difference when combined.

ABMS Component Recipes

SL9 (trace minerals)

1L	Component
800 mL	diH₂O
12.8 g	Nitrilotriacetic acid
2 g	FeSO₄•7H₂O (note: first dissolve in 2M HCl)
104 mg	CoCl₂ (anhydrous)
122 mg	MnCl₂•4H₂O
70 mg	ZnCl₂
36 mg	Na₂MoO₄•2H₂O
13 mg	NiCl₂

Add 1 mL of 10 mL 10× solution:

60 mg	H₃BO₃
20 mg	CuCl₂•2H₂O

To dissolve all components, adjust pH to ~7 or slightly higher using 1 M NaOH while you bring the volume to 1000 mL with diH₂O. Note: this requires a lot of NaOH, so leave room when bringing up to volume.

Mineral Solution (20×)

500 mL	Component
10 g	NH₄Cl
5.8 g	MgSO₄•7H₂O
1 g	KNO₃
0.67 g	CaCl₂•2H₂O †
20 mg	(NH₄)₆Mo₇O₂₄•4H₂O
10 mL	SL 9

† Substitution: You may use 465.8 µL of a 1 M CaCl₂ per 500 ml of stock solution for this component. The final concentration of Ca²⁺ in the 20× stock is 9.12 mM.

Bring to 500mL in a graduated cylinder with deionized water.

Phosphate Buffer (20×)

500mL	Components
68 g	KH₂PO₄
132.5 g	Na₂HPO₄•H₂O

Bring to 500 mL with diH₂O; pH should be ~6.9-7.0 (you may add 400 mL diH₂O first and bring pH up to 6.8 then complete volume to 500mL)

* Source: Averhoff lab

Included topic: ProtocolsRecipesABMS

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)

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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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

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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

Add dH₂O to full volume in an appropriately-sized flask. Combine the following, making sure each component fully dissolves before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl
1 ml	6 ml	5% Antifoam*
16 g	48 g	Agar*

ANTIFOAM: Open the bottle under sterile conditions (near a flame) only, and continue agitating after the addition of the 5% antifoam.

AGAR: Measure 8 grams of agar per 500 ml bottle and add to each individual bottle before aliquoting the LB. Autoclave to homogenize.

Each liter of LB Agar makes 2 bottles of 500 ml.

Notes and variations:

Common synonyms are Luria-Broth and Luria-Bertani medium.
This is the "Miller" formulation of LB with 10 g NaCl per liter (1% w/v). Another common formulation has only 5 g NaCl per liter (0.5% w/v)! They are NOT interchangeable.
If you are performing sacB counter-selection, you do not want to include NaCl in your agar. Use the above recipe, but swap out 5-10% w/v (50-100g per liter) sucrose in place of any NaCl. Sucrose can be directly combined with the other components and autoclaved (no need to filter sterilize it separately).
To make "soft" or "top" agar for phage studies, use only 7-8 g agar per liter.

Included topic: ProtocolsRecipesLysogenyBrothAgar

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

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, Na₂HPO_{4 (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)

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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:

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.
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

MOB: Mobility Media

This media is useful for enhancing the mobility of ADP1, which moves across the plate by "twitching".

1L	Final [ ]	Component
5 g	0.5%	agarose
5 g	2.0%	tryptone
2.5 g	10 mM	NaCl

After pouring your gel, let it set overnight at room temperature. The next day, spot with 1µl of your overnight-grown culture, leave the plate to dry for 15 minutes at room temperature, wrap in Parafilm, invert, and incubate at 30°C. You should see spreading over the next 24 hours.

Included topic: ProtocolsMobilityMedia

Blood - Heart Infusion Agar

Heart Infusion Agar supplemented with sterile Sheep's Blood. Used for the cultivation of fastidious organisms (Bee gut microbiome species et al)

1L	Component
44 g	Heart Infusion Agar (HIA) (Difco brand)
50mL	Sterile Sheep's Blood

Autoclave water and media (without blood) on a 20min liquid cycle. Transfer autoclaved media to hot water bath at 55 deg Celsius. Allow to equilibrate for 30mins to 1hr. Add 100mL blood (using PipetAid) and stir to mix (a stir bar isn't necessary, but do not shake). Smooth, small circles while the bottle is on a flat surface is sufficient to mix the blood and warm agar. Add necessary antibiotics and supplements (remember the volume is now 1100 mL, not 1 L) and pour into plates.

As with other agar, after autoclaving the HIA you can let it solidify at room temperature. After microwaving to dissolve, it still needs to be cooled before adding blood.

Included topic: ProtocolsRecipesBloodHeartInfusion

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.

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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.

Add dH₂O to desired volume in an appropriate flask. Combine the following, making sure the components completely dissolve before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl

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. For 1L bottles, add 500 ml of LB to each. Each liter of LB makes 2 bottles. Autoclave.

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.

0.5L	1L	4.5L	5L	Component	MW
2.67 g	5.34 g	24.03 g	26.7 g	Potassium Phosphate (dibasic) K₂HPO₄	MW 174.18
1 g	2 g	9 g	10 g	Potassium Phosphate (monobasic) KH₂PO₄	MW 136.07
0.5 g	1 g	4.5 g	5 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.25 g	0.5 g	2.25 g	2.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to final volume and autoclave.

Note: 4.5 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 Heptahydrate MgSO₄•7H₂O (separately autoclaved stock)
0.5 ml	550 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)
0.406 mM	Magnesium (Mg²⁺)
8.41 mM	Sulfate (SO₄^2-)
45.3 mM	Phosphate (PO₄^3-)
1.70 mM	Citrate
139 µM	Glucose (in DM25)

Note: Recipe was corrected on 2026-04-22 to use MgSO₄•7H₂O.

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

Included topic: ProtocolsRecipesM9Minimal

ABMS: Acinetobacter Minimal Succinate

Courtesy of the Averhoff lab, with modifications for available reagents.

To make standard ABMS, combine the following pre-sterilized ingredients at room temperature:

1L	5L	Component
880 mL	4.4 L	diH₂O
20 mL	100 mL	1M Sodium Succinate
50 mL	250 mL	20× Mineral Solution
50 mL	250 mL	20× Phosphate Buffer

To make ABMS agar:

Per liter of media, add 16 g/L agar and 1 mL/L antifoam to diH₂O, then autoclave in a 2 L flask. When water and agar are cool enough to touch, add component solutions. Optional: warm components at 55°C in a water bath before combining with agar to reduce the temperature difference when combined.

ABMS Component Recipes

SL9 (trace minerals)

1L	Component
800 mL	diH₂O
12.8 g	Nitrilotriacetic acid
2 g	FeSO₄•7H₂O (note: first dissolve in 2M HCl)
104 mg	CoCl₂ (anhydrous)
122 mg	MnCl₂•4H₂O
70 mg	ZnCl₂
36 mg	Na₂MoO₄•2H₂O
13 mg	NiCl₂

Add 1 mL of 10 mL 10× solution:

60 mg	H₃BO₃
20 mg	CuCl₂•2H₂O

To dissolve all components, adjust pH to ~7 or slightly higher using 1 M NaOH while you bring the volume to 1000 mL with diH₂O. Note: this requires a lot of NaOH, so leave room when bringing up to volume.

Mineral Solution (20×)

500 mL	Component
10 g	NH₄Cl
5.8 g	MgSO₄•7H₂O
1 g	KNO₃
0.67 g	CaCl₂•2H₂O †
20 mg	(NH₄)₆Mo₇O₂₄•4H₂O
10 mL	SL 9

† Substitution: You may use 465.8 µL of a 1 M CaCl₂ per 500 ml of stock solution for this component. The final concentration of Ca²⁺ in the 20× stock is 9.12 mM.

Bring to 500mL in a graduated cylinder with deionized water.

Phosphate Buffer (20×)

500mL	Components
68 g	KH₂PO₄
132.5 g	Na₂HPO₄•H₂O

Bring to 500 mL with diH₂O; pH should be ~6.9-7.0 (you may add 400 mL diH₂O first and bring pH up to 6.8 then complete volume to 500mL)

* Source: Averhoff lab

Included topic: ProtocolsRecipesABMS

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

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

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

Add dH₂O to full volume in an appropriately-sized flask. Combine the following, making sure each component fully dissolves before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl
1 ml	6 ml	5% Antifoam*
16 g	48 g	Agar*

ANTIFOAM: Open the bottle under sterile conditions (near a flame) only, and continue agitating after the addition of the 5% antifoam.

AGAR: Measure 8 grams of agar per 500 ml bottle and add to each individual bottle before aliquoting the LB. Autoclave to homogenize.

Each liter of LB Agar makes 2 bottles of 500 ml.

Notes and variations:

Common synonyms are Luria-Broth and Luria-Bertani medium.
This is the "Miller" formulation of LB with 10 g NaCl per liter (1% w/v). Another common formulation has only 5 g NaCl per liter (0.5% w/v)! They are NOT interchangeable.
If you are performing sacB counter-selection, you do not want to include NaCl in your agar. Use the above recipe, but swap out 5-10% w/v (50-100g per liter) sucrose in place of any NaCl. Sucrose can be directly combined with the other components and autoclaved (no need to filter sterilize it separately).
To make "soft" or "top" agar for phage studies, use only 7-8 g agar per liter.

Included topic: ProtocolsRecipesLysogenyBrothAgar

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

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, Na₂HPO_{4 (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: ProtocolsRecipesM9

Warning: Can't find topic Lab.ProtocolsRecipesSOBplates

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:

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.
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

MOB: Mobility Media

This media is useful for enhancing the mobility of ADP1, which moves across the plate by "twitching".

1L	Final [ ]	Component
5 g	0.5%	agarose
5 g	2.0%	tryptone
2.5 g	10 mM	NaCl

After pouring your gel, let it set overnight at room temperature. The next day, spot with 1µl of your overnight-grown culture, leave the plate to dry for 15 minutes at room temperature, wrap in Parafilm, invert, and incubate at 30°C. You should see spreading over the next 24 hours.

Included topic: ProtocolsMobilityMedia

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Blood - Heart Infusion Agar

Heart Infusion Agar supplemented with sterile Sheep's Blood. Used for the cultivation of fastidious organisms (Bee gut microbiome species et al)

1L	Component
44 g	Heart Infusion Agar (HIA) (Difco brand)
50mL	Sterile Sheep's Blood

Autoclave water and media (without blood) on a 20min liquid cycle. Transfer autoclaved media to hot water bath at 55 deg Celsius. Allow to equilibrate for 30mins to 1hr. Add 100mL blood (using PipetAid) and stir to mix (a stir bar isn't necessary, but do not shake). Smooth, small circles while the bottle is on a flat surface is sufficient to mix the blood and warm agar. Add necessary antibiotics and supplements (remember the volume is now 1100 mL, not 1 L) and pour into plates.

As with other agar, after autoclaving the HIA you can let it solidify at room temperature. After microwaving to dissolve, it still needs to be cooled before adding blood.

Included topic: ProtocolsRecipesBloodHeartInfusion

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

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Revision 392016-12-15 - JuliePerreau

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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.

Add dH₂O to desired volume in an appropriate flask. Combine the following, making sure the components completely dissolve before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl

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. For 1L bottles, add 500 ml of LB to each. Each liter of LB makes 2 bottles. Autoclave.

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.

0.5L	1L	4.5L	5L	Component	MW
2.67 g	5.34 g	24.03 g	26.7 g	Potassium Phosphate (dibasic) K₂HPO₄	MW 174.18
1 g	2 g	9 g	10 g	Potassium Phosphate (monobasic) KH₂PO₄	MW 136.07
0.5 g	1 g	4.5 g	5 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.25 g	0.5 g	2.25 g	2.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to final volume and autoclave.

Note: 4.5 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 Heptahydrate MgSO₄•7H₂O (separately autoclaved stock)
0.5 ml	550 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)
0.406 mM	Magnesium (Mg²⁺)
8.41 mM	Sulfate (SO₄^2-)
45.3 mM	Phosphate (PO₄^3-)
1.70 mM	Citrate
139 µM	Glucose (in DM25)

Note: Recipe was corrected on 2026-04-22 to use MgSO₄•7H₂O.

Included topic: ProtocolsRecipesDavisMingioli

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

Included topic: ProtocolsRecipesM9Minimal

ABMS: Acinetobacter Minimal Succinate

Courtesy of the Averhoff lab, with modifications for available reagents.

To make standard ABMS, combine the following pre-sterilized ingredients at room temperature:

1L	5L	Component
880 mL	4.4 L	diH₂O
20 mL	100 mL	1M Sodium Succinate
50 mL	250 mL	20× Mineral Solution
50 mL	250 mL	20× Phosphate Buffer

To make ABMS agar:

Per liter of media, add 16 g/L agar and 1 mL/L antifoam to diH₂O, then autoclave in a 2 L flask. When water and agar are cool enough to touch, add component solutions. Optional: warm components at 55°C in a water bath before combining with agar to reduce the temperature difference when combined.

ABMS Component Recipes

SL9 (trace minerals)

1L	Component
800 mL	diH₂O
12.8 g	Nitrilotriacetic acid
2 g	FeSO₄•7H₂O (note: first dissolve in 2M HCl)
104 mg	CoCl₂ (anhydrous)
122 mg	MnCl₂•4H₂O
70 mg	ZnCl₂
36 mg	Na₂MoO₄•2H₂O
13 mg	NiCl₂

Add 1 mL of 10 mL 10× solution:

60 mg	H₃BO₃
20 mg	CuCl₂•2H₂O

To dissolve all components, adjust pH to ~7 or slightly higher using 1 M NaOH while you bring the volume to 1000 mL with diH₂O. Note: this requires a lot of NaOH, so leave room when bringing up to volume.

Mineral Solution (20×)

500 mL	Component
10 g	NH₄Cl
5.8 g	MgSO₄•7H₂O
1 g	KNO₃
0.67 g	CaCl₂•2H₂O †
20 mg	(NH₄)₆Mo₇O₂₄•4H₂O
10 mL	SL 9

† Substitution: You may use 465.8 µL of a 1 M CaCl₂ per 500 ml of stock solution for this component. The final concentration of Ca²⁺ in the 20× stock is 9.12 mM.

Bring to 500mL in a graduated cylinder with deionized water.

Phosphate Buffer (20×)

500mL	Components
68 g	KH₂PO₄
132.5 g	Na₂HPO₄•H₂O

Bring to 500 mL with diH₂O; pH should be ~6.9-7.0 (you may add 400 mL diH₂O first and bring pH up to 6.8 then complete volume to 500mL)

* Source: Averhoff lab

Included topic: ProtocolsRecipesABMS

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

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

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

Add dH₂O to full volume in an appropriately-sized flask. Combine the following, making sure each component fully dissolves before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl
1 ml	6 ml	5% Antifoam*
16 g	48 g	Agar*

ANTIFOAM: Open the bottle under sterile conditions (near a flame) only, and continue agitating after the addition of the 5% antifoam.

AGAR: Measure 8 grams of agar per 500 ml bottle and add to each individual bottle before aliquoting the LB. Autoclave to homogenize.

Each liter of LB Agar makes 2 bottles of 500 ml.

Notes and variations:

Common synonyms are Luria-Broth and Luria-Bertani medium.
This is the "Miller" formulation of LB with 10 g NaCl per liter (1% w/v). Another common formulation has only 5 g NaCl per liter (0.5% w/v)! They are NOT interchangeable.
If you are performing sacB counter-selection, you do not want to include NaCl in your agar. Use the above recipe, but swap out 5-10% w/v (50-100g per liter) sucrose in place of any NaCl. Sucrose can be directly combined with the other components and autoclaved (no need to filter sterilize it separately).
To make "soft" or "top" agar for phage studies, use only 7-8 g agar per liter.

Included topic: ProtocolsRecipesLysogenyBrothAgar

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

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, Na₂HPO_{4 (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: ProtocolsRecipesM9

Warning: Can't find topic Lab.ProtocolsRecipesSOBplates

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:

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.
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

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MOB: Mobility Media

This media is useful for enhancing the mobility of ADP1, which moves across the plate by "twitching".

1L	Final [ ]	Component
5 g	0.5%	agarose
5 g	2.0%	tryptone
2.5 g	10 mM	NaCl

After pouring your gel, let it set overnight at room temperature. The next day, spot with 1µl of your overnight-grown culture, leave the plate to dry for 15 minutes at room temperature, wrap in Parafilm, invert, and incubate at 30°C. You should see spreading over the next 24 hours.

Included topic: ProtocolsMobilityMedia

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

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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.

Add dH₂O to desired volume in an appropriate flask. Combine the following, making sure the components completely dissolve before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl

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. For 1L bottles, add 500 ml of LB to each. Each liter of LB makes 2 bottles. Autoclave.

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.

0.5L	1L	4.5L	5L	Component	MW
2.67 g	5.34 g	24.03 g	26.7 g	Potassium Phosphate (dibasic) K₂HPO₄	MW 174.18
1 g	2 g	9 g	10 g	Potassium Phosphate (monobasic) KH₂PO₄	MW 136.07
0.5 g	1 g	4.5 g	5 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.25 g	0.5 g	2.25 g	2.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to final volume and autoclave.

Note: 4.5 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 Heptahydrate MgSO₄•7H₂O (separately autoclaved stock)
0.5 ml	550 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)
0.406 mM	Magnesium (Mg²⁺)
8.41 mM	Sulfate (SO₄^2-)
45.3 mM	Phosphate (PO₄^3-)
1.70 mM	Citrate
139 µM	Glucose (in DM25)

Note: Recipe was corrected on 2026-04-22 to use MgSO₄•7H₂O.

Included topic: ProtocolsRecipesDavisMingioli

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

Included topic: ProtocolsRecipesM9Minimal

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ABMS: Acinetobacter Minimal Succinate

Courtesy of the Averhoff lab, with modifications for available reagents.

To make standard ABMS, combine the following pre-sterilized ingredients at room temperature:

1L	5L	Component
880 mL	4.4 L	diH₂O
20 mL	100 mL	1M Sodium Succinate
50 mL	250 mL	20× Mineral Solution
50 mL	250 mL	20× Phosphate Buffer

To make ABMS agar:

Per liter of media, add 16 g/L agar and 1 mL/L antifoam to diH₂O, then autoclave in a 2 L flask. When water and agar are cool enough to touch, add component solutions. Optional: warm components at 55°C in a water bath before combining with agar to reduce the temperature difference when combined.

ABMS Component Recipes

SL9 (trace minerals)

1L	Component
800 mL	diH₂O
12.8 g	Nitrilotriacetic acid
2 g	FeSO₄•7H₂O (note: first dissolve in 2M HCl)
104 mg	CoCl₂ (anhydrous)
122 mg	MnCl₂•4H₂O
70 mg	ZnCl₂
36 mg	Na₂MoO₄•2H₂O
13 mg	NiCl₂

Add 1 mL of 10 mL 10× solution:

60 mg	H₃BO₃
20 mg	CuCl₂•2H₂O

To dissolve all components, adjust pH to ~7 or slightly higher using 1 M NaOH while you bring the volume to 1000 mL with diH₂O. Note: this requires a lot of NaOH, so leave room when bringing up to volume.

Mineral Solution (20×)

500 mL	Component
10 g	NH₄Cl
5.8 g	MgSO₄•7H₂O
1 g	KNO₃
0.67 g	CaCl₂•2H₂O †
20 mg	(NH₄)₆Mo₇O₂₄•4H₂O
10 mL	SL 9

† Substitution: You may use 465.8 µL of a 1 M CaCl₂ per 500 ml of stock solution for this component. The final concentration of Ca²⁺ in the 20× stock is 9.12 mM.

Bring to 500mL in a graduated cylinder with deionized water.

Phosphate Buffer (20×)

500mL	Components
68 g	KH₂PO₄
132.5 g	Na₂HPO₄•H₂O

Bring to 500 mL with diH₂O; pH should be ~6.9-7.0 (you may add 400 mL diH₂O first and bring pH up to 6.8 then complete volume to 500mL)

* Source: Averhoff lab

Included topic: ProtocolsRecipesABMS

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

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

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

Add dH₂O to full volume in an appropriately-sized flask. Combine the following, making sure each component fully dissolves before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl
1 ml	6 ml	5% Antifoam*
16 g	48 g	Agar*

ANTIFOAM: Open the bottle under sterile conditions (near a flame) only, and continue agitating after the addition of the 5% antifoam.

AGAR: Measure 8 grams of agar per 500 ml bottle and add to each individual bottle before aliquoting the LB. Autoclave to homogenize.

Each liter of LB Agar makes 2 bottles of 500 ml.

Notes and variations:

Common synonyms are Luria-Broth and Luria-Bertani medium.
This is the "Miller" formulation of LB with 10 g NaCl per liter (1% w/v). Another common formulation has only 5 g NaCl per liter (0.5% w/v)! They are NOT interchangeable.
If you are performing sacB counter-selection, you do not want to include NaCl in your agar. Use the above recipe, but swap out 5-10% w/v (50-100g per liter) sucrose in place of any NaCl. Sucrose can be directly combined with the other components and autoclaved (no need to filter sterilize it separately).
To make "soft" or "top" agar for phage studies, use only 7-8 g agar per liter.

Included topic: ProtocolsRecipesLysogenyBrothAgar

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

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, Na₂HPO_{4 (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: ProtocolsRecipesM9

Warning: Can't find topic Lab.ProtocolsRecipesSOBplates

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:

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.
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

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Revision 372015-10-22 - JeffreyBarrick

META TOPICPARENT	name="ProtocolsMediaRecipes"

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.

Add dH₂O to desired volume in an appropriate flask. Combine the following, making sure the components completely dissolve before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl

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. For 1L bottles, add 500 ml of LB to each. Each liter of LB makes 2 bottles. Autoclave.

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.

0.5L	1L	4.5L	5L	Component	MW
2.67 g	5.34 g	24.03 g	26.7 g	Potassium Phosphate (dibasic) K₂HPO₄	MW 174.18
1 g	2 g	9 g	10 g	Potassium Phosphate (monobasic) KH₂PO₄	MW 136.07
0.5 g	1 g	4.5 g	5 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.25 g	0.5 g	2.25 g	2.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to final volume and autoclave.

Note: 4.5 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 Heptahydrate MgSO₄•7H₂O (separately autoclaved stock)
0.5 ml	550 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)
0.406 mM	Magnesium (Mg²⁺)
8.41 mM	Sulfate (SO₄^2-)
45.3 mM	Phosphate (PO₄^3-)
1.70 mM	Citrate
139 µM	Glucose (in DM25)

Note: Recipe was corrected on 2026-04-22 to use MgSO₄•7H₂O.

Included topic: ProtocolsRecipesDavisMingioli

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

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

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

Add dH₂O to full volume in an appropriately-sized flask. Combine the following, making sure each component fully dissolves before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl
1 ml	6 ml	5% Antifoam*
16 g	48 g	Agar*

ANTIFOAM: Open the bottle under sterile conditions (near a flame) only, and continue agitating after the addition of the 5% antifoam.

AGAR: Measure 8 grams of agar per 500 ml bottle and add to each individual bottle before aliquoting the LB. Autoclave to homogenize.

Each liter of LB Agar makes 2 bottles of 500 ml.

Notes and variations:

Common synonyms are Luria-Broth and Luria-Bertani medium.
This is the "Miller" formulation of LB with 10 g NaCl per liter (1% w/v). Another common formulation has only 5 g NaCl per liter (0.5% w/v)! They are NOT interchangeable.
If you are performing sacB counter-selection, you do not want to include NaCl in your agar. Use the above recipe, but swap out 5-10% w/v (50-100g per liter) sucrose in place of any NaCl. Sucrose can be directly combined with the other components and autoclaved (no need to filter sterilize it separately).
To make "soft" or "top" agar for phage studies, use only 7-8 g agar per liter.

Included topic: ProtocolsRecipesLysogenyBrothAgar

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

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, Na₂HPO_{4 (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: ProtocolsRecipesM9

Warning: Can't find topic Lab.ProtocolsRecipesSOBplates

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:

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.
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

Deleted:

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Special Media

Included topic: ProtocolsRecipesSpecialMedia

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Revision 362015-09-20 - JordanMonk

META TOPICPARENT	name="ProtocolsMediaRecipes"

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.

Add dH₂O to desired volume in an appropriate flask. Combine the following, making sure the components completely dissolve before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl

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. For 1L bottles, add 500 ml of LB to each. Each liter of LB makes 2 bottles. Autoclave.

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.

0.5L	1L	4.5L	5L	Component	MW
2.67 g	5.34 g	24.03 g	26.7 g	Potassium Phosphate (dibasic) K₂HPO₄	MW 174.18
1 g	2 g	9 g	10 g	Potassium Phosphate (monobasic) KH₂PO₄	MW 136.07
0.5 g	1 g	4.5 g	5 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.25 g	0.5 g	2.25 g	2.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to final volume and autoclave.

Note: 4.5 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 Heptahydrate MgSO₄•7H₂O (separately autoclaved stock)
0.5 ml	550 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)
0.406 mM	Magnesium (Mg²⁺)
8.41 mM	Sulfate (SO₄^2-)
45.3 mM	Phosphate (PO₄^3-)
1.70 mM	Citrate
139 µM	Glucose (in DM25)

Note: Recipe was corrected on 2026-04-22 to use MgSO₄•7H₂O.

Included topic: ProtocolsRecipesDavisMingioli

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

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

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

Add dH₂O to full volume in an appropriately-sized flask. Combine the following, making sure each component fully dissolves before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl
1 ml	6 ml	5% Antifoam*
16 g	48 g	Agar*

ANTIFOAM: Open the bottle under sterile conditions (near a flame) only, and continue agitating after the addition of the 5% antifoam.

AGAR: Measure 8 grams of agar per 500 ml bottle and add to each individual bottle before aliquoting the LB. Autoclave to homogenize.

Each liter of LB Agar makes 2 bottles of 500 ml.

Notes and variations:

Common synonyms are Luria-Broth and Luria-Bertani medium.
This is the "Miller" formulation of LB with 10 g NaCl per liter (1% w/v). Another common formulation has only 5 g NaCl per liter (0.5% w/v)! They are NOT interchangeable.
If you are performing sacB counter-selection, you do not want to include NaCl in your agar. Use the above recipe, but swap out 5-10% w/v (50-100g per liter) sucrose in place of any NaCl. Sucrose can be directly combined with the other components and autoclaved (no need to filter sterilize it separately).
To make "soft" or "top" agar for phage studies, use only 7-8 g agar per liter.

Included topic: ProtocolsRecipesLysogenyBrothAgar

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

Changed:

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Warning: Can't find topic Lab.ProtocolsRecipesSOBplates

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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, Na₂HPO_{4 (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: ProtocolsRecipesM9

Changed:

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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, Na₂HPO_{4 (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)

>
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Warning: Can't find topic Lab.ProtocolsRecipesSOBplates

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:

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.
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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Revision 352015-06-23 - JeffreyBarrick

META TOPICPARENT	name="ProtocolsMediaRecipes"

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

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

Add dH₂O to desired volume in an appropriate flask. Combine the following, making sure the components completely dissolve before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl

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. For 1L bottles, add 500 ml of LB to each. Each liter of LB makes 2 bottles. Autoclave.

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!)

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Included topic: ProtocolsRecipesLysogenyBroth

DM: Davis-Mingioli

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

0.5L	1L	4.5L	5L	Component	MW
2.67 g	5.34 g	24.03 g	26.7 g	Potassium Phosphate (dibasic) K₂HPO₄	MW 174.18
1 g	2 g	9 g	10 g	Potassium Phosphate (monobasic) KH₂PO₄	MW 136.07
0.5 g	1 g	4.5 g	5 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.25 g	0.5 g	2.25 g	2.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to final volume and autoclave.

Note: 4.5 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 Heptahydrate MgSO₄•7H₂O (separately autoclaved stock)
0.5 ml	550 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)
0.406 mM	Magnesium (Mg²⁺)
8.41 mM	Sulfate (SO₄^2-)
45.3 mM	Phosphate (PO₄^3-)
1.70 mM	Citrate
139 µM	Glucose (in DM25)

Note: Recipe was corrected on 2026-04-22 to use MgSO₄•7H₂O.

Included topic: ProtocolsRecipesDavisMingioli

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

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

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

Add dH₂O to full volume in an appropriately-sized flask. Combine the following, making sure each component fully dissolves before adding the next:

1L	6L	Component
10 g	60 g	Tryptone
5 g	30 g	Yeast Extract
10 g	60 g	NaCl
1 ml	6 ml	5% Antifoam*
16 g	48 g	Agar*

ANTIFOAM: Open the bottle under sterile conditions (near a flame) only, and continue agitating after the addition of the 5% antifoam.

AGAR: Measure 8 grams of agar per 500 ml bottle and add to each individual bottle before aliquoting the LB. Autoclave to homogenize.

Each liter of LB Agar makes 2 bottles of 500 ml.

Notes and variations:

Common synonyms are Luria-Broth and Luria-Bertani medium.
This is the "Miller" formulation of LB with 10 g NaCl per liter (1% w/v). Another common formulation has only 5 g NaCl per liter (0.5% w/v)! They are NOT interchangeable.
If you are performing sacB counter-selection, you do not want to include NaCl in your agar. Use the above recipe, but swap out 5-10% w/v (50-100g per liter) sucrose in place of any NaCl. Sucrose can be directly combined with the other components and autoclaved (no need to filter sterilize it separately).
To make "soft" or "top" agar for phage studies, use only 7-8 g agar per liter.

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Included topic: ProtocolsRecipesLysogenyBrothAgar

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

Warning: Can't find topic Lab.ProtocolsRecipesSOBplates

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, Na₂HPO_{4 (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:

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.
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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attachment="Acinetobacter_Minimal_Succinate_Media_Recipe.docx" attr="" comment="Minimal Succinate Media for culturing Acinetobacter baylyi ADP1" date="1401826885" name="Acinetobacter_Minimal_Succinate_Media_Recipe.docx" path="Acinetobacter Minimal Succinate Media Recipe.docx" size="87008" stream="Acinetobacter Minimal Succinate Media Recipe.docx" tmpFilename="/usr/tmp/CGItemp40964" user="BrianRenda" version="1"

Revision 342014-06-03 - BrianRenda

META TOPICPARENT	name="ProtocolsMediaRecipes"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

DM: Davis-Mingioli

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

0.5L	1L	4.5L	5L	Component	MW
2.67 g	5.34 g	24.03 g	26.7 g	Potassium Phosphate (dibasic) K₂HPO₄	MW 174.18
1 g	2 g	9 g	10 g	Potassium Phosphate (monobasic) KH₂PO₄	MW 136.07
0.5 g	1 g	4.5 g	5 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.25 g	0.5 g	2.25 g	2.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to final volume and autoclave.

Note: 4.5 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 Heptahydrate MgSO₄•7H₂O (separately autoclaved stock)
0.5 ml	550 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)
0.406 mM	Magnesium (Mg²⁺)
8.41 mM	Sulfate (SO₄^2-)
45.3 mM	Phosphate (PO₄^3-)
1.70 mM	Citrate
139 µM	Glucose (in DM25)

Note: Recipe was corrected on 2026-04-22 to use MgSO₄•7H₂O.

Included topic: ProtocolsRecipesDavisMingioli

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

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

Deleted:

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<

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 (SO₄^2-)
106.1 mM	Phosphate (PO₄^3-)
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

Warning: Can't find topic Lab.ProtocolsRecipesLuria-Broth

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

Warning: Can't find topic Lab.ProtocolsRecipesSOBplates

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, Na₂HPO_{4 (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:

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.
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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Revision 332014-04-11 - CraigBarnhart

META TOPICPARENT	name="ProtocolsMediaRecipes"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

DM: Davis-Mingioli

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

0.5L	1L	4.5L	5L	Component	MW
2.67 g	5.34 g	24.03 g	26.7 g	Potassium Phosphate (dibasic) K₂HPO₄	MW 174.18
1 g	2 g	9 g	10 g	Potassium Phosphate (monobasic) KH₂PO₄	MW 136.07
0.5 g	1 g	4.5 g	5 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.25 g	0.5 g	2.25 g	2.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to final volume and autoclave.

Note: 4.5 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 Heptahydrate MgSO₄•7H₂O (separately autoclaved stock)
0.5 ml	550 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)
0.406 mM	Magnesium (Mg²⁺)
8.41 mM	Sulfate (SO₄^2-)
45.3 mM	Phosphate (PO₄^3-)
1.70 mM	Citrate
139 µM	Glucose (in DM25)

Note: Recipe was corrected on 2026-04-22 to use MgSO₄•7H₂O.

Included topic: ProtocolsRecipesDavisMingioli

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

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 (SO₄^2-)
106.1 mM	Phosphate (PO₄^3-)
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

Warning: Can't find topic Lab.ProtocolsRecipesLuria-Broth

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

Warning: Can't find topic Lab.ProtocolsRecipesSOBplates

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, Na₂HPO_{4 (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:

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.
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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Revision 322013-09-05 - GabrielSuarez

META TOPICPARENT	name="ProtocolsMediaRecipes"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

DM: Davis-Mingioli

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

0.5L	1L	4.5L	5L	Component	MW
2.67 g	5.34 g	24.03 g	26.7 g	Potassium Phosphate (dibasic) K₂HPO₄	MW 174.18
1 g	2 g	9 g	10 g	Potassium Phosphate (monobasic) KH₂PO₄	MW 136.07
0.5 g	1 g	4.5 g	5 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.25 g	0.5 g	2.25 g	2.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to final volume and autoclave.

Note: 4.5 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 Heptahydrate MgSO₄•7H₂O (separately autoclaved stock)
0.5 ml	550 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)
0.406 mM	Magnesium (Mg²⁺)
8.41 mM	Sulfate (SO₄^2-)
45.3 mM	Phosphate (PO₄^3-)
1.70 mM	Citrate
139 µM	Glucose (in DM25)

Note: Recipe was corrected on 2026-04-22 to use MgSO₄•7H₂O.

Included topic: ProtocolsRecipesDavisMingioli

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

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 (SO₄^2-)
106.1 mM	Phosphate (PO₄^3-)
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

Warning: Can't find topic Lab.ProtocolsRecipesLuria-Broth

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

Warning: Can't find topic Lab.ProtocolsRecipesSOBplates

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, Na₂HPO_{4 (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:

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.
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

META FILEATTACHMENT	attachment="Media_Recipes.docx" attr="" comment="" date="1303834127" name="Media_Recipes.docx" path="Media Recipes.docx" size="15424" stream="Media Recipes.docx" tmpFilename="/usr/tmp/CGItemp19503" user="BrianRenda" version="1"

Revision 312013-08-21 - CraigBarnhart

META TOPICPARENT	name="ProtocolsMediaRecipes"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

DM: Davis-Mingioli

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

0.5L	1L	4.5L	5L	Component	MW
2.67 g	5.34 g	24.03 g	26.7 g	Potassium Phosphate (dibasic) K₂HPO₄	MW 174.18
1 g	2 g	9 g	10 g	Potassium Phosphate (monobasic) KH₂PO₄	MW 136.07
0.5 g	1 g	4.5 g	5 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.25 g	0.5 g	2.25 g	2.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to final volume and autoclave.

Note: 4.5 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 Heptahydrate MgSO₄•7H₂O (separately autoclaved stock)
0.5 ml	550 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)
0.406 mM	Magnesium (Mg²⁺)
8.41 mM	Sulfate (SO₄^2-)
45.3 mM	Phosphate (PO₄^3-)
1.70 mM	Citrate
139 µM	Glucose (in DM25)

Note: Recipe was corrected on 2026-04-22 to use MgSO₄•7H₂O.

Included topic: ProtocolsRecipesDavisMingioli

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

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 (SO₄^2-)
106.1 mM	Phosphate (PO₄^3-)
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

Warning: Can't find topic Lab.ProtocolsRecipesLuria-Broth

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

Warning: Can't find topic Lab.ProtocolsRecipesSOBplates

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, Na₂HPO_{4 (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:

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.
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

Added:

>
>

Special Media

Included topic: ProtocolsRecipesSpecialMedia

META FILEATTACHMENT	attachment="Media_Recipes.docx" attr="" comment="" date="1303834127" name="Media_Recipes.docx" path="Media Recipes.docx" size="15424" stream="Media Recipes.docx" tmpFilename="/usr/tmp/CGItemp19503" user="BrianRenda" version="1"

Revision 302013-06-24 - CraigBarnhart

META TOPICPARENT	name="ProtocolsMediaRecipes"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

DM: Davis-Mingioli

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

0.5L	1L	4.5L	5L	Component	MW
2.67 g	5.34 g	24.03 g	26.7 g	Potassium Phosphate (dibasic) K₂HPO₄	MW 174.18
1 g	2 g	9 g	10 g	Potassium Phosphate (monobasic) KH₂PO₄	MW 136.07
0.5 g	1 g	4.5 g	5 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.25 g	0.5 g	2.25 g	2.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to final volume and autoclave.

Note: 4.5 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 Heptahydrate MgSO₄•7H₂O (separately autoclaved stock)
0.5 ml	550 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)
0.406 mM	Magnesium (Mg²⁺)
8.41 mM	Sulfate (SO₄^2-)
45.3 mM	Phosphate (PO₄^3-)
1.70 mM	Citrate
139 µM	Glucose (in DM25)

Note: Recipe was corrected on 2026-04-22 to use MgSO₄•7H₂O.

Included topic: ProtocolsRecipesDavisMingioli

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

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 (SO₄^2-)
106.1 mM	Phosphate (PO₄^3-)
50.0 mM	Lactic Acid

-- Main.GabrielSuarez - 05 Sep 2013

Included topic:ProtocolsRecipesS2

Added:

>
>

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

Warning: Can't find topic Lab.ProtocolsRecipesLuria-Broth

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

Warning: Can't find topic Lab.ProtocolsRecipesSOBplates

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, Na₂HPO_{4 (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:

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.
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

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Revision 292013-06-03 - AurkoDasgupta

META TOPICPARENT	name="ProtocolsMediaRecipes"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

DM: Davis-Mingioli

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

0.5L	1L	4.5L	5L	Component	MW
2.67 g	5.34 g	24.03 g	26.7 g	Potassium Phosphate (dibasic) K₂HPO₄	MW 174.18
1 g	2 g	9 g	10 g	Potassium Phosphate (monobasic) KH₂PO₄	MW 136.07
0.5 g	1 g	4.5 g	5 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.25 g	0.5 g	2.25 g	2.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to final volume and autoclave.

Note: 4.5 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 Heptahydrate MgSO₄•7H₂O (separately autoclaved stock)
0.5 ml	550 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)
0.406 mM	Magnesium (Mg²⁺)
8.41 mM	Sulfate (SO₄^2-)
45.3 mM	Phosphate (PO₄^3-)
1.70 mM	Citrate
139 µM	Glucose (in DM25)

Note: Recipe was corrected on 2026-04-22 to use MgSO₄•7H₂O.

Included topic: ProtocolsRecipesDavisMingioli

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

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

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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 (SO₄^2-)
106.1 mM	Phosphate (PO₄^3-)
50.0 mM	Lactic Acid

-- Main.GabrielSuarez - 05 Sep 2013

Included topic:ProtocolsRecipesS2

Solid Media

Warning: Can't find topic Lab.ProtocolsRecipesLuria-Broth

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

Warning: Can't find topic Lab.ProtocolsRecipesSOBplates

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, Na₂HPO_{4 (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:

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.
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

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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

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DM: Davis-Mingioli

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

0.5L	1L	4.5L	5L	Component	MW
2.67 g	5.34 g	24.03 g	26.7 g	Potassium Phosphate (dibasic) K₂HPO₄	MW 174.18
1 g	2 g	9 g	10 g	Potassium Phosphate (monobasic) KH₂PO₄	MW 136.07
0.5 g	1 g	4.5 g	5 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.25 g	0.5 g	2.25 g	2.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to final volume and autoclave.

Note: 4.5 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 Heptahydrate MgSO₄•7H₂O (separately autoclaved stock)
0.5 ml	550 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)
0.406 mM	Magnesium (Mg²⁺)
8.41 mM	Sulfate (SO₄^2-)
45.3 mM	Phosphate (PO₄^3-)
1.70 mM	Citrate
139 µM	Glucose (in DM25)

Note: Recipe was corrected on 2026-04-22 to use MgSO₄•7H₂O.

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Included topic: ProtocolsRecipesDavisMinimal

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Included topic: ProtocolsRecipesDavisMingioli

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

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

Solid Media

Warning: Can't find topic Lab.ProtocolsRecipesLuria-Broth

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

Warning: Can't find topic Lab.ProtocolsRecipesSOBplates

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, Na₂HPO_{4 (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:

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.
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

META FILEATTACHMENT	attachment="Media_Recipes.docx" attr="" comment="" date="1303834127" name="Media_Recipes.docx" path="Media Recipes.docx" size="15424" stream="Media Recipes.docx" tmpFilename="/usr/tmp/CGItemp19503" user="BrianRenda" version="1"

Revision 272012-08-01 - CraigBarnhart

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

Warning: Can't find topic Lab.ProtocolsRecipesDavisMinimal

Included topic: ProtocolsRecipesDavisMinimal

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

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

Added:

>
>

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

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

Added:

>
>

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

Solid Media

Warning: Can't find topic Lab.ProtocolsRecipesLuria-Broth

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

Warning: Can't find topic Lab.ProtocolsRecipesSOBplates

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, Na₂HPO_{4 (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:

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.
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

META FILEATTACHMENT	attachment="Media_Recipes.docx" attr="" comment="" date="1303834127" name="Media_Recipes.docx" path="Media Recipes.docx" size="15424" stream="Media Recipes.docx" tmpFilename="/usr/tmp/CGItemp19503" user="BrianRenda" version="1"

Revision 262012-06-21 - AurkoDasgupta

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

Warning: Can't find topic Lab.ProtocolsRecipesDavisMinimal

Included topic: ProtocolsRecipesDavisMinimal

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

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

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

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

Solid Media

Warning: Can't find topic Lab.ProtocolsRecipesLuria-Broth

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

Warning: Can't find topic Lab.ProtocolsRecipesSOBplates

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, Na₂HPO_{4 (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:

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.
Miller J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Included topic: ProtocolsPapillationAgar

Added:

>
>

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

Deleted:

<
<

META FILEATTACHMENT	attachment="Media_Recipes.docx" attr="" comment="" date="1303834127" name="Media_Recipes.docx" path="Media Recipes.docx" size="15424" stream="Media Recipes.docx" tmpFilename="/usr/tmp/CGItemp19503" user="BrianRenda" version="1"

Revision 252012-06-06 - CraigBarnhart

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

Warning: Can't find topic Lab.ProtocolsRecipesDavisMinimal

Included topic: ProtocolsRecipesDavisMinimal

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

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

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

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

Solid Media

Warning: Can't find topic Lab.ProtocolsRecipesLuria-Broth

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

Warning: Can't find topic Lab.ProtocolsRecipesSOBplates

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, Na₂HPO_{4 (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:

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.
Miller J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Included topic: ProtocolsPapillationAgar

Added:

>
>

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

META FILEATTACHMENT	attachment="Media_Recipes.docx" attr="" comment="" date="1303834127" name="Media_Recipes.docx" path="Media Recipes.docx" size="15424" stream="Media Recipes.docx" tmpFilename="/usr/tmp/CGItemp19503" user="BrianRenda" version="1"

Revision 242012-06-04 - CraigBarnhart

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

Warning: Can't find topic Lab.ProtocolsRecipesDavisMinimal

Included topic: ProtocolsRecipesDavisMinimal

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

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

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

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

Solid Media

Warning: Can't find topic Lab.ProtocolsRecipesLuria-Broth

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

Warning: Can't find topic Lab.ProtocolsRecipesSOBplates

Added:

>
>

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, Na₂HPO_{4 (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:

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.
Miller J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Included topic: ProtocolsPapillationAgar

META FILEATTACHMENT	attachment="Media_Recipes.docx" attr="" comment="" date="1303834127" name="Media_Recipes.docx" path="Media Recipes.docx" size="15424" stream="Media Recipes.docx" tmpFilename="/usr/tmp/CGItemp19503" user="BrianRenda" version="1"

Revision 232012-05-04 - JeffreyBarrick

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

Warning: Can't find topic Lab.ProtocolsRecipesDavisMinimal

Included topic: ProtocolsRecipesDavisMinimal

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

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

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

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

Solid Media

Warning: Can't find topic Lab.ProtocolsRecipesLuria-Broth

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

Warning: Can't find topic Lab.ProtocolsRecipesSOBplates

Included topic: ProtocolsRecipesSOBplates

Added:

>
>

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:

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.
Miller J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Included topic: ProtocolsPapillationAgar

META FILEATTACHMENT	attachment="Media_Recipes.docx" attr="" comment="" date="1303834127" name="Media_Recipes.docx" path="Media Recipes.docx" size="15424" stream="Media Recipes.docx" tmpFilename="/usr/tmp/CGItemp19503" user="BrianRenda" version="1"

Revision 222012-05-02 - CraigBarnhart

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

Warning: Can't find topic Lab.ProtocolsRecipesDavisMinimal

Included topic: ProtocolsRecipesDavisMinimal

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

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

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

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

Solid Media

Warning: Can't find topic Lab.ProtocolsRecipesLuria-Broth

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

Warning: Can't find topic Lab.ProtocolsRecipesSOBplates

Included topic: ProtocolsRecipesSOBplates

META FILEATTACHMENT	attachment="Media_Recipes.docx" attr="" comment="" date="1303834127" name="Media_Recipes.docx" path="Media Recipes.docx" size="15424" stream="Media Recipes.docx" tmpFilename="/usr/tmp/CGItemp19503" user="BrianRenda" version="1"

Revision 212012-01-11 - CraigBarnhart

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

Warning: Can't find topic Lab.ProtocolsRecipesDavisMinimal

Included topic: ProtocolsRecipesDavisMinimal

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

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

Added:

>
>

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 two-dram 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.

Containers for SOB/SOC

Autoclave the two-dram vials of SOC in the plastic container and store in the cardboard container when dry. Make sure lids are unscrewed prior to autoclaving and screwed shut afterward. The plastic container can have the rack popped out (see image on the right) after autoclaving to dry any water that accumulated at the bottom of the box and avoid the growth of mold and such.

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

Solid Media

Warning: Can't find topic Lab.ProtocolsRecipesLuria-Broth

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

Added:

>
>

Warning: Can't find topic Lab.ProtocolsRecipesSOBplates

Included topic: ProtocolsRecipesSOBplates

META FILEATTACHMENT	attachment="Media_Recipes.docx" attr="" comment="" date="1303834127" name="Media_Recipes.docx" path="Media Recipes.docx" size="15424" stream="Media Recipes.docx" tmpFilename="/usr/tmp/CGItemp19503" user="BrianRenda" version="1"

Revision 202011-10-05 - JeffreyBarrick

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

Warning: Can't find topic Lab.ProtocolsRecipesDavisMinimal

Included topic: ProtocolsRecipesDavisMinimal

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

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

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

Solid Media

Warning: Can't find topic Lab.ProtocolsRecipesLuria-Broth

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

Changed:

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<

Warning: Can't find topic Lab.ProtocolsRecipesDM0

>
>

MC: Minimal Citrate

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

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

Changed:

<
<

Included topic: ProtocolsRecipesDM0+

>
>

Included topic: ProtocolsRecipesMinimalCitrate

META FILEATTACHMENT	attachment="Media_Recipes.docx" attr="" comment="" date="1303834127" name="Media_Recipes.docx" path="Media Recipes.docx" size="15424" stream="Media Recipes.docx" tmpFilename="/usr/tmp/CGItemp19503" user="BrianRenda" version="1"

Revision 192011-08-30 - CraigBarnhart

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

Warning: Can't find topic Lab.ProtocolsRecipesDavisMinimal

Included topic: ProtocolsRecipesDavisMinimal

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

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

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

Solid Media

Warning: Can't find topic Lab.ProtocolsRecipesLuria-Broth

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

Added:

>
>

Warning: Can't find topic Lab.ProtocolsRecipesDM0

Included topic: ProtocolsRecipesDM0+

META FILEATTACHMENT	attachment="Media_Recipes.docx" attr="" comment="" date="1303834127" name="Media_Recipes.docx" path="Media Recipes.docx" size="15424" stream="Media Recipes.docx" tmpFilename="/usr/tmp/CGItemp19503" user="BrianRenda" version="1"

Revision 182011-07-29 - CraigBarnhart

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

Warning: Can't find topic Lab.ProtocolsRecipesDavisMinimal

Included topic: ProtocolsRecipesDavisMinimal

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

Included topic: ProtocolsRecipesM9Minimal

Added:

>
>

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

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

Added:

>
>

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

Solid Media

Warning: Can't find topic Lab.ProtocolsRecipesLuria-Broth

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

Changed:

<
<

Warning: Can't find topic Lab.ProtocolsRecipesDavisMinimal

>
>

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

Changed:

<
<

Included topic: ProtocolsRecipesDavisMinimal

>
>

Included topic: ProtocolsRecipesMinimalGlucose

META FILEATTACHMENT	attachment="Media_Recipes.docx" attr="" comment="" date="1303834127" name="Media_Recipes.docx" path="Media Recipes.docx" size="15424" stream="Media Recipes.docx" tmpFilename="/usr/tmp/CGItemp19503" user="BrianRenda" version="1"

Revision 172011-07-11 - CraigBarnhart

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

Warning: Can't find topic Lab.ProtocolsRecipesDavisMinimal

Included topic: ProtocolsRecipesDavisMinimal

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

Included topic: ProtocolsRecipesM9Minimal

Warning: Can't find topic Lab.ProtocolsRecipesSOC

Included topic: ProtocolsRecipesSOC

Added:

>
>

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

Solid Media

Warning: Can't find topic Lab.ProtocolsRecipesLuria-Broth

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

Warning: Can't find topic Lab.ProtocolsRecipesDavisMinimal

Included topic: ProtocolsRecipesDavisMinimal

META FILEATTACHMENT	attachment="Media_Recipes.docx" attr="" comment="" date="1303834127" name="Media_Recipes.docx" path="Media Recipes.docx" size="15424" stream="Media Recipes.docx" tmpFilename="/usr/tmp/CGItemp19503" user="BrianRenda" version="1"

Revision 162011-06-14 - CraigBarnhart

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

Changed:

<
<

DM: Davis Minimal

>
>

Warning: Can't find topic Lab.ProtocolsRecipesDavisMinimal

Changed:

<
<

7 g

Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ (H₂O)₃

MW 228.17

>
>

Included topic: ProtocolsRecipesDavisMinimal

Deleted:

<
<

5 g	Potassium Phosphate (monobasic) H₂KPO₄	MW 136.07
1 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to 1 liter. Autoclave. Then add

1 ml	10% Magnesium Sulfate MgSO₄	MW 120.37	Note* The MgSO₄ must be autoclaved separately.
1 ml	0.2% Thiamin (vitamin B1)

Changed:

<
<

Final nutrient concentrations:

>
>

Warning: Can't find topic Lab.ProtocolsRecipesNZYBroth

Deleted:

<
<

67.4 mM	Phosphate (PO₄^3-)
8.38 mM	Sulfate (SO₄^2-)
15.1 mM	Ammonium (NH₄)
5.1 mM	Sodium (Na⁺)
98.1 mM	Potassium (K⁺)
0.83 mM	Magnesium (MGgsup>2+)
1.7 mM	Citrate
139 µM	Glucose (in DM25)

Changed:

<
<

NZY+ Broth

>
>

Included topic: ProtocolsRecipesNZY+Broth

Changed:

<
<

10 g	NZ Amine (casein hydrolysate)

>
>

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 (SO₄^2-)
42.2 mM	Phosphate (PO₄^3-)

Deleted:

<
<

5 g	Yeast Extract
5 g	NaCl

Changed:

<
<

Add dH₂O to 950 ml. Adjust pH to 7.5 using NaOH (Requires ~350 µl of 10 N). Autoclave. Then add.

>
>

Included topic: ProtocolsRecipesM9Minimal

Deleted:

<
<

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

M9 Minimal

Make 5× M9 salts:

64 g	Sodium phosphate (dibasic, 7 hydrate) Na₂HPO₄•H₂O
15 g	Potassium phosphate (monobasic) KH₂PO₄
2.5 g	Sodium chloride, NaCl
5 g	Ammonium chloride NH₄Cl

Add dH₂O to 1 liter. Autoclave.

Combine the following ingredients (all pre-sterilized):

200 ml	5× M9 salts
2 ml	1M Magnesium sulfate MgSO₄
0.1 ml	1 M Calcium chloride CaCl₂
40 ml	10% (w/v) glucose (or other carbon source)

Add sterile dH₂O to 1 liter. Autoclave.

Warning: Can't find topic Lab.ProtocolsRecipesSOC

Included topic: ProtocolsRecipesSOC

Solid Media

Changed:

<
<

LB: Luria-Broth

>
>

Warning: Can't find topic Lab.ProtocolsRecipesLuria-Broth

Changed:

<
<

Combine in a 2 L flask:

>
>

Included topic: ProtocolsRecipesLuria-Broth

Deleted:

<
<

15 g	Tryptone
7.5 g	Yeast Extract
15 g	NaCl
24 g	Agar
1.5 ml	5% Antifoam	or	.75 ml	10% Antifoam

Changed:

<
<

Add dH₂O to 1.5 L. Autoclave.

>
>

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

Changed:

<
<

Note: This is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

>
>

Included topic: ProtocolsRecipesTetrazoliumSugar

Changed:

<
<

To make "soft" agar for phage studies, use only 7 g agar per liter.

>
>

Warning: Can't find topic Lab.ProtocolsRecipesDavisMinimal

Changed:

<
<

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

>
>

Included topic: ProtocolsRecipesDavisMinimal

Deleted:

<
<

Combine in a 2 L flask:

15 g	Tryptone
1.5 g	Yeast Extract
7.5 g	NaCl
24 g	Agar
1.5 ml	5% Antifoam	or	.75 ml	10% Antifoam

Add dH₂O to 1.3 liter.

Combine in a 500 ml flask:

6 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:

1.5 ml

TTC (5%)

or

7.5 ml

TTC (1%)

DM: Davis Minimal (MG, MA, MM, ML, ...)

10.5 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ H₂O
3 g	Potassium Phosphate (monobasic) H₂KPO₄
1.5 g	Ammonium Sulfate (NH₄)₂ SO₄
0.75 g	Sodium Citrate (tribasic, dihydrate) (Na₃Citrate 2•H₂O₄

Add dH₂O to 0.8 liter in a 2 L flask.

24 g	Agar
1.5 ml	5% Antifoam	or	.75 ml	10% Antifoam

Add dH₂O to 0.5 liter in a 1 L flask.

6 g	Sugar (glucose = MG, arabinose = MA, maltose = MM, lactose = ML, ...

Add dH₂O to 0.2 liter in a 500 ml flask.

Autoclave. Important! Do not combine solutions before autoclaving. Agar and phosphate, phosphate and sugar, and agar and sugar react with each other at high temperatures to create compounds that inhibit bacterial cell growth.

Combine three flasks and add

1.5 ml	10% Magnesium Sulfate MgSO₄
1.5 ml	0.2% Thiamin (vitamin B1)

Total volume is 1.5 L, which makes 60-80 plates.

META FILEATTACHMENT	attachment="Media_Recipes.docx" attr="" comment="" date="1303834127" name="Media_Recipes.docx" path="Media Recipes.docx" size="15424" stream="Media Recipes.docx" tmpFilename="/usr/tmp/CGItemp19503" user="BrianRenda" version="1"

Revision 152011-06-11 - JeffreyBarrick

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

DM: Davis Minimal

7 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ (H₂O)₃	MW 228.17
5 g	Potassium Phosphate (monobasic) H₂KPO₄	MW 136.07
1 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to 1 liter. Autoclave. Then add

1 ml	10% Magnesium Sulfate MgSO₄	MW 120.37	Note* The MgSO₄ must be autoclaved separately.
1 ml	0.2% Thiamin (vitamin B1)

Final nutrient concentrations:

67.4 mM	Phosphate (PO₄^3-)
8.38 mM	Sulfate (SO₄^2-)
15.1 mM	Ammonium (NH₄)
5.1 mM	Sodium (Na⁺)
98.1 mM	Potassium (K⁺)
0.83 mM	Magnesium (MGgsup>2+)
1.7 mM	Citrate
139 µM	Glucose (in DM25)

NZY+ Broth

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

Add dH₂O to 950 ml. Adjust pH to 7.5 using NaOH (Requires ~350 µl of 10 N). Autoclave. Then add.

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

M9 Minimal

Make 5× M9 salts:

64 g	Sodium phosphate (dibasic, 7 hydrate) Na₂HPO₄•H₂O
15 g	Potassium phosphate (monobasic) KH₂PO₄
2.5 g	Sodium chloride, NaCl
5 g	Ammonium chloride NH₄Cl

Add dH₂O to 1 liter. Autoclave.

Combine the following ingredients (all pre-sterilized):

200 ml	5× M9 salts
2 ml	1M Magnesium sulfate MgSO₄
0.1 ml	1 M Calcium chloride CaCl₂
40 ml	10% (w/v) glucose (or other carbon source)

Add sterile dH₂O to 1 liter. Autoclave.

Changed:

<
<

Warning: Can't find topic Lab.ProtocolsRecipesLuriaSOC

>
>

Warning: Can't find topic Lab.ProtocolsRecipesSOC

Included topic: ProtocolsRecipesSOC

Solid Media

LB: Luria-Broth

Combine in a 2 L flask:

15 g	Tryptone
7.5 g	Yeast Extract
15 g	NaCl
24 g	Agar
1.5 ml	5% Antifoam	or	.75 ml	10% Antifoam

Add dH₂O to 1.5 L. Autoclave.

Note: This is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

To make "soft" agar for phage studies, use only 7 g agar per liter.

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

Combine in a 2 L flask:

15 g	Tryptone
1.5 g	Yeast Extract
7.5 g	NaCl
24 g	Agar
1.5 ml	5% Antifoam	or	.75 ml	10% Antifoam

Add dH₂O to 1.3 liter.

Combine in a 500 ml flask:

6 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:

1.5 ml

TTC (5%)

or

7.5 ml

TTC (1%)

DM: Davis Minimal (MG, MA, MM, ML, ...)

10.5 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ H₂O
3 g	Potassium Phosphate (monobasic) H₂KPO₄

Changed:

<
<

1.5 g	Ammonium Sulfate (NH₄)₂ SO₄
0.75 g	Sodium Citrate (tribasic, dihydrate) (Na₃Citrate 2•H₂O₄

>
>

1.5 g	Ammonium Sulfate (NH₄)₂ SO₄
0.75 g	Sodium Citrate (tribasic, dihydrate) (Na₃Citrate 2•H₂O₄

Add dH₂O to 0.8 liter in a 2 L flask.

Changed:

<
<

24 g / 10 g

Agar

>
>

24 g

Agar

1.5 ml

5% Antifoam

or

.75 ml

10% Antifoam

Add dH₂O to 0.5 liter in a 1 L flask.

6 g	Sugar (glucose = MG, arabinose = MA, maltose = MM, lactose = ML, ...

Add dH₂O to 0.2 liter in a 500 ml flask.

Autoclave. Important! Do not combine solutions before autoclaving. Agar and phosphate, phosphate and sugar, and agar and sugar react with each other at high temperatures to create compounds that inhibit bacterial cell growth.

Combine three flasks and add

1.5 ml	10% Magnesium Sulfate MgSO₄
1.5 ml	0.2% Thiamin (vitamin B1)

Total volume is 1.5 L, which makes 60-80 plates.

META FILEATTACHMENT	attachment="Media_Recipes.docx" attr="" comment="" date="1303834127" name="Media_Recipes.docx" path="Media Recipes.docx" size="15424" stream="Media Recipes.docx" tmpFilename="/usr/tmp/CGItemp19503" user="BrianRenda" version="1"

Revision 142011-05-31 - CraigBarnhart

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

DM: Davis Minimal

7 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ (H₂O)₃	MW 228.17
5 g	Potassium Phosphate (monobasic) H₂KPO₄	MW 136.07
1 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to 1 liter. Autoclave. Then add

1 ml	10% Magnesium Sulfate MgSO₄	MW 120.37	Note* The MgSO₄ must be autoclaved separately.
1 ml	0.2% Thiamin (vitamin B1)

Final nutrient concentrations:

67.4 mM	Phosphate (PO₄^3-)
8.38 mM	Sulfate (SO₄^2-)
15.1 mM	Ammonium (NH₄)
5.1 mM	Sodium (Na⁺)
98.1 mM	Potassium (K⁺)
0.83 mM	Magnesium (MGgsup>2+)
1.7 mM	Citrate
139 µM	Glucose (in DM25)

NZY+ Broth

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

Add dH₂O to 950 ml. Adjust pH to 7.5 using NaOH (Requires ~350 µl of 10 N). Autoclave. Then add.

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

M9 Minimal

Make 5× M9 salts:

64 g	Sodium phosphate (dibasic, 7 hydrate) Na₂HPO₄•H₂O
15 g	Potassium phosphate (monobasic) KH₂PO₄
2.5 g	Sodium chloride, NaCl
5 g	Ammonium chloride NH₄Cl

Add dH₂O to 1 liter. Autoclave.

Combine the following ingredients (all pre-sterilized):

200 ml	5× M9 salts
2 ml	1M Magnesium sulfate MgSO₄
0.1 ml	1 M Calcium chloride CaCl₂
40 ml	10% (w/v) glucose (or other carbon source)

Add sterile dH₂O to 1 liter. Autoclave.

Warning: Can't find topic Lab.ProtocolsRecipesLuriaSOC

Included topic: ProtocolsRecipesSOC

Solid Media

LB: Luria-Broth

Combine in a 2 L flask:

15 g	Tryptone
7.5 g	Yeast Extract
15 g	NaCl
24 g	Agar
1.5 ml	5% Antifoam	or	.75 ml	10% Antifoam

Add dH₂O to 1.5 L. Autoclave.

Note: This is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

To make "soft" agar for phage studies, use only 7 g agar per liter.

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

Combine in a 2 L flask:

15 g	Tryptone
1.5 g	Yeast Extract
7.5 g	NaCl
24 g	Agar
1.5 ml	5% Antifoam	or	.75 ml	10% Antifoam

Add dH₂O to 1.3 liter.

Combine in a 500 ml flask:

6 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:

1.5 ml

TTC (5%)

or

7.5 ml

TTC (1%)

DM: Davis Minimal (MG, MA, MM, ML, ...)

10.5 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ H₂O
3 g	Potassium Phosphate (monobasic) H₂KPO₄
1.5 g	Ammonium Sulfate (NH₄)₂ SO₄
0.75 g	Sodium Citrate (tribasic, dihydrate) (Na₃Citrate 2•H₂O₄

Add dH₂O to 0.8 liter in a 2 L flask.

24 g / 10 g	Agar
1.5 ml	5% Antifoam	or	.75 ml	10% Antifoam

Add dH₂O to 0.5 liter in a 1 L flask.

6 g	Sugar (glucose = MG, arabinose = MA, maltose = MM, lactose = ML, ...

Add dH₂O to 0.2 liter in a 500 ml flask.

Autoclave. Important! Do not combine solutions before autoclaving. Agar and phosphate, phosphate and sugar, and agar and sugar react with each other at high temperatures to create compounds that inhibit bacterial cell growth.

Combine three flasks and add

1.5 ml	10% Magnesium Sulfate MgSO₄
1.5 ml	0.2% Thiamin (vitamin B1)

Total volume is 1.5 L, which makes 60-80 plates.

META FILEATTACHMENT	attachment="Media_Recipes.docx" attr="" comment="" date="1303834127" name="Media_Recipes.docx" path="Media Recipes.docx" size="15424" stream="Media Recipes.docx" tmpFilename="/usr/tmp/CGItemp19503" user="BrianRenda" version="1"

Revision 132011-05-19 - JeffreyBarrick

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

DM: Davis Minimal

7 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ (H₂O)₃	MW 228.17
5 g	Potassium Phosphate (monobasic) H₂KPO₄	MW 136.07
1 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to 1 liter. Autoclave. Then add

1 ml	10% Magnesium Sulfate MgSO₄	MW 120.37	Note* The MgSO₄ must be autoclaved separately.
1 ml	0.2% Thiamin (vitamin B1)

Final nutrient concentrations:

67.4 mM	Phosphate (PO₄^3-)
8.38 mM	Sulfate (SO₄^2-)
15.1 mM	Ammonium (NH₄)
5.1 mM	Sodium (Na⁺)
98.1 mM	Potassium (K⁺)
0.83 mM	Magnesium (MGgsup>2+)
1.7 mM	Citrate
139 µM	Glucose (in DM25)

NZY+ Broth

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

Add dH₂O to 950 ml. Adjust pH to 7.5 using NaOH (Requires ~350 µl of 10 N). Autoclave. Then add.

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

M9 Minimal

Make 5× M9 salts:

64 g	Sodium phosphate (dibasic, 7 hydrate) Na₂HPO₄•H₂O
15 g	Potassium phosphate (monobasic) KH₂PO₄
2.5 g	Sodium chloride, NaCl
5 g	Ammonium chloride NH₄Cl

Add dH₂O to 1 liter. Autoclave.

Combine the following ingredients (all pre-sterilized):

200 ml	5× M9 salts
2 ml	1M Magnesium sulfate MgSO₄
0.1 ml	1 M Calcium chloride CaCl₂
40 ml	10% (w/v) glucose (or other carbon source)

Add sterile dH₂O to 1 liter. Autoclave.

Added:

>
>

Warning: Can't find topic Lab.ProtocolsRecipesLuriaSOC

Included topic: ProtocolsRecipesSOC

Solid Media

LB: Luria-Broth

Combine in a 2 L flask:

15 g	Tryptone
7.5 g	Yeast Extract
15 g	NaCl
24 g	Agar
1.5 ml	5% Antifoam	or	.75 ml	10% Antifoam

Add dH₂O to 1.5 L. Autoclave.

Note: This is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

To make "soft" agar for phage studies, use only 7 g agar per liter.

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

Combine in a 2 L flask:

15 g	Tryptone
1.5 g	Yeast Extract
7.5 g	NaCl
24 g	Agar
1.5 ml	5% Antifoam	or	.75 ml	10% Antifoam

Add dH₂O to 1.3 liter.

Combine in a 500 ml flask:

6 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:

1.5 ml

TTC (5%)

or

7.5 ml

TTC (1%)

DM: Davis Minimal (MG, MA, MM, ML, ...)

10.5 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ H₂O
3 g	Potassium Phosphate (monobasic) H₂KPO₄
1.5 g	Ammonium Sulfate (NH₄)₂ SO₄
0.75 g	Sodium Citrate (tribasic, dihydrate) (Na₃Citrate 2•H₂O₄

Add dH₂O to 0.8 liter in a 2 L flask.

24 g / 10 g	Agar
1.5 ml	5% Antifoam	or	.75 ml	10% Antifoam

Add dH₂O to 0.5 liter in a 1 L flask.

6 g	Sugar (glucose = MG, arabinose = MA, maltose = MM, lactose = ML, ...

Add dH₂O to 0.2 liter in a 500 ml flask.

Autoclave. Important! Do not combine solutions before autoclaving. Agar and phosphate, phosphate and sugar, and agar and sugar react with each other at high temperatures to create compounds that inhibit bacterial cell growth.

Combine three flasks and add

1.5 ml	10% Magnesium Sulfate MgSO₄
1.5 ml	0.2% Thiamin (vitamin B1)

Total volume is 1.5 L, which makes 60-80 plates.

Added:

>
>

META FILEATTACHMENT	attachment="Media_Recipes.docx" attr="" comment="" date="1303834127" name="Media_Recipes.docx" path="Media Recipes.docx" size="15424" stream="Media Recipes.docx" tmpFilename="/usr/tmp/CGItemp19503" user="BrianRenda" version="1"

Revision 122011-04-26 - BrianRenda

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

DM: Davis Minimal

7 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ (H₂O)₃	MW 228.17
5 g	Potassium Phosphate (monobasic) H₂KPO₄	MW 136.07
1 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to 1 liter. Autoclave. Then add

1 ml	10% Magnesium Sulfate MgSO₄	MW 120.37	Note* The MgSO₄ must be autoclaved separately.
1 ml	0.2% Thiamin (vitamin B1)

Final nutrient concentrations:

67.4 mM	Phosphate (PO₄^3-)
8.38 mM	Sulfate (SO₄^2-)
15.1 mM	Ammonium (NH₄)
5.1 mM	Sodium (Na⁺)
98.1 mM	Potassium (K⁺)
0.83 mM	Magnesium (MGgsup>2+)
1.7 mM	Citrate
139 µM	Glucose (in DM25)

NZY+ Broth

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

Add dH₂O to 950 ml. Adjust pH to 7.5 using NaOH (Requires ~350 µl of 10 N). Autoclave. Then add.

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

M9 Minimal

Make 5× M9 salts:

64 g	Sodium phosphate (dibasic, 7 hydrate) Na₂HPO₄•H₂O
15 g	Potassium phosphate (monobasic) KH₂PO₄
2.5 g	Sodium chloride, NaCl
5 g	Ammonium chloride NH₄Cl

Add dH₂O to 1 liter. Autoclave.

Combine the following ingredients (all pre-sterilized):

200 ml	5× M9 salts
2 ml	1M Magnesium sulfate MgSO₄
0.1 ml	1 M Calcium chloride CaCl₂
40 ml	10% (w/v) glucose (or other carbon source)

Add sterile dH₂O to 1 liter. Autoclave.

Solid Media

LB: Luria-Broth

Combine in a 2 L flask:

15 g	Tryptone
7.5 g	Yeast Extract
15 g	NaCl
24 g	Agar
1.5 ml	5% Antifoam	or	.75 ml	10% Antifoam

Add dH₂O to 1.5 L. Autoclave.

Note: This is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

To make "soft" agar for phage studies, use only 7 g agar per liter.

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

Combine in a 2 L flask:

15 g	Tryptone
1.5 g	Yeast Extract
7.5 g	NaCl
24 g	Agar
1.5 ml	5% Antifoam	or	.75 ml	10% Antifoam

Add dH₂O to 1.3 liter.

Combine in a 500 ml flask:

6 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:

1.5 ml

TTC (5%)

or

7.5 ml

TTC (1%)

DM: Davis Minimal (MG, MA, MM, ML, ...)

10.5 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ H₂O
3 g	Potassium Phosphate (monobasic) H₂KPO₄
1.5 g	Ammonium Sulfate (NH₄)₂ SO₄
0.75 g	Sodium Citrate (tribasic, dihydrate) (Na₃Citrate 2•H₂O₄

Add dH₂O to 0.8 liter in a 2 L flask.

24 g / 10 g	Agar
1.5 ml	5% Antifoam	or	.75 ml	10% Antifoam

Add dH₂O to 0.5 liter in a 1 L flask.

6 g	Sugar (glucose = MG, arabinose = MA, maltose = MM, lactose = ML, ...

Add dH₂O to 0.2 liter in a 500 ml flask.

Autoclave. Important! Do not combine solutions before autoclaving. Agar and phosphate, phosphate and sugar, and agar and sugar react with each other at high temperatures to create compounds that inhibit bacterial cell growth.

Combine three flasks and add

1.5 ml	10% Magnesium Sulfate MgSO₄
1.5 ml	0.2% Thiamin (vitamin B1)

Total volume is 1.5 L, which makes 60-80 plates.

Added:

>
>

META FILEATTACHMENT	attachment="Media_Recipes.docx" attr="" comment="" date="1303834127" name="Media_Recipes.docx" path="Media Recipes.docx" size="15424" stream="Media Recipes.docx" tmpFilename="/usr/tmp/CGItemp19503" user="BrianRenda" version="1"

Revision 112011-03-24 - CraigBarnhart

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

DM: Davis Minimal

7 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ (H₂O)₃	MW 228.17
5 g	Potassium Phosphate (monobasic) H₂KPO₄	MW 136.07
1 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to 1 liter. Autoclave. Then add

1 ml	10% Magnesium Sulfate MgSO₄	MW 120.37	Note* The MgSO₄ must be autoclaved separately.
1 ml	0.2% Thiamin (vitamin B1)

Final nutrient concentrations:

67.4 mM	Phosphate (PO₄^3-)
8.38 mM	Sulfate (SO₄^2-)
15.1 mM	Ammonium (NH₄)
5.1 mM	Sodium (Na⁺)
98.1 mM	Potassium (K⁺)
0.83 mM	Magnesium (MGgsup>2+)
1.7 mM	Citrate
139 µM	Glucose (in DM25)

NZY+ Broth

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

Add dH₂O to 950 ml. Adjust pH to 7.5 using NaOH (Requires ~350 µl of 10 N). Autoclave. Then add.

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

M9 Minimal

Make 5× M9 salts:

64 g	Sodium phosphate (dibasic, 7 hydrate) Na₂HPO₄•H₂O
15 g	Potassium phosphate (monobasic) KH₂PO₄
2.5 g	Sodium chloride, NaCl
5 g	Ammonium chloride NH₄Cl

Add dH₂O to 1 liter. Autoclave.

Combine the following ingredients (all pre-sterilized):

200 ml	5× M9 salts
2 ml	1M Magnesium sulfate MgSO₄
0.1 ml	1 M Calcium chloride CaCl₂
40 ml	10% (w/v) glucose (or other carbon source)

Add sterile dH₂O to 1 liter. Autoclave.

Solid Media

LB: Luria-Broth

Combine in a 2 L flask:

15 g	Tryptone
7.5 g	Yeast Extract
15 g	NaCl
24 g	Agar

Changed:

<
<

1.5 ml

5% Antifoam

>
>

1.5 ml

5% Antifoam

or

.75 ml

10% Antifoam

Add dH₂O to 1.5 L. Autoclave.

Note: This is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

To make "soft" agar for phage studies, use only 7 g agar per liter.

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

Combine in a 2 L flask:

15 g	Tryptone
1.5 g	Yeast Extract
7.5 g	NaCl
24 g	Agar

Changed:

<
<

1.5 ml

5% Antifoam

>
>

1.5 ml

5% Antifoam

or

.75 ml

10% Antifoam

Add dH₂O to 1.3 liter.

Combine in a 500 ml flask:

6 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:

Changed:

<
<

1.5 ml

TTC (5%)

>
>

1.5 ml

TTC (5%)

or

7.5 ml

TTC (1%)

Added:

>
>

DM: Davis Minimal (MG, MA, MM, ML, ...)

10.5 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ H₂O
3 g	Potassium Phosphate (monobasic) H₂KPO₄
1.5 g	Ammonium Sulfate (NH₄)₂ SO₄
0.75 g	Sodium Citrate (tribasic, dihydrate) (Na₃Citrate 2•H₂O₄

Add dH₂O to 0.8 liter in a 2 L flask.

Changed:

<
<

24 g / 10 g	Agar
1.5 ml	5% Antifoam

>
>

24 g / 10 g	Agar
1.5 ml	5% Antifoam	or	.75 ml	10% Antifoam

Add dH₂O to 0.5 liter in a 1 L flask.

6 g	Sugar (glucose = MG, arabinose = MA, maltose = MM, lactose = ML, ...

Add dH₂O to 0.2 liter in a 500 ml flask.

Autoclave. Important! Do not combine solutions before autoclaving. Agar and phosphate, phosphate and sugar, and agar and sugar react with each other at high temperatures to create compounds that inhibit bacterial cell growth.

Combine three flasks and add

1.5 ml	10% Magnesium Sulfate MgSO₄
1.5 ml	0.2% Thiamin (vitamin B1)

Total volume is 1.5 L, which makes 60-80 plates.

Revision 102011-03-24 - CraigBarnhart

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

DM: Davis Minimal

7 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ (H₂O)₃	MW 228.17
5 g	Potassium Phosphate (monobasic) H₂KPO₄	MW 136.07
1 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to 1 liter. Autoclave. Then add

1 ml	10% Magnesium Sulfate MgSO₄	MW 120.37	Note* The MgSO₄ must be autoclaved separately.
1 ml	0.2% Thiamin (vitamin B1)

Final nutrient concentrations:

67.4 mM	Phosphate (PO₄^3-)
8.38 mM	Sulfate (SO₄^2-)
15.1 mM	Ammonium (NH₄)
5.1 mM	Sodium (Na⁺)
98.1 mM	Potassium (K⁺)
0.83 mM	Magnesium (MGgsup>2+)
1.7 mM	Citrate
139 µM	Glucose (in DM25)

NZY+ Broth

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

Add dH₂O to 950 ml. Adjust pH to 7.5 using NaOH (Requires ~350 µl of 10 N). Autoclave. Then add.

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

M9 Minimal

Make 5× M9 salts:

64 g	Sodium phosphate (dibasic, 7 hydrate) Na₂HPO₄•H₂O
15 g	Potassium phosphate (monobasic) KH₂PO₄
2.5 g	Sodium chloride, NaCl
5 g	Ammonium chloride NH₄Cl

Add dH₂O to 1 liter. Autoclave.

Combine the following ingredients (all pre-sterilized):

200 ml	5× M9 salts
2 ml	1M Magnesium sulfate MgSO₄
0.1 ml	1 M Calcium chloride CaCl₂
40 ml	10% (w/v) glucose (or other carbon source)

Add sterile dH₂O to 1 liter. Autoclave.

Solid Media

LB: Luria-Broth

Combine in a 2 L flask:

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.

Note: This is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

To make "soft" agar for phage studies, use only 7 g agar per liter.

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

Combine in a 2 L flask:

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:

6 g	Sugar (arabinose = TA, maltose = TM, lactose = TL)

Changed:

<
<

Add dH₂O to 1.3 liter.

>
>

Add dH₂O to 0.2 liter.

Autoclave sugar and media separately.

Added:

>
>

Total combined volume is 1.5 liters.

Combine and add:

1.5 ml

TTC (5%)

DM: Davis Minimal (MG, MA, MM, ML, ...)

10.5 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ H₂O
3 g	Potassium Phosphate (monobasic) H₂KPO₄
1.5 g	Ammonium Sulfate (NH₄)₂ SO₄
0.75 g	Sodium Citrate (tribasic, dihydrate) (Na₃Citrate 2•H₂O₄

Add dH₂O to 0.8 liter in a 2 L flask.

24 g / 10 g	Agar
1.5 ml	5% Antifoam

Add dH₂O to 0.5 liter in a 1 L flask.

6 g	Sugar (glucose = MG, arabinose = MA, maltose = MM, lactose = ML, ...

Add dH₂O to 0.2 liter in a 500 ml flask.

Autoclave. Important! Do not combine solutions before autoclaving. Agar and phosphate, phosphate and sugar, and agar and sugar react with each other at high temperatures to create compounds that inhibit bacterial cell growth.

Combine three flasks and add

1.5 ml	10% Magnesium Sulfate MgSO₄
1.5 ml	0.2% Thiamin (vitamin B1)

Total volume is 1.5 L, which makes 60-80 plates.

Revision 92011-03-20 - JeffreyBarrick

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

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

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Included topic: ProtocolsRecipesLuriaBertani

DM: Davis Minimal

7 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ (H₂O)₃	MW 228.17
5 g	Potassium Phosphate (monobasic) H₂KPO₄	MW 136.07
1 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to 1 liter. Autoclave. Then add

1 ml	10% Magnesium Sulfate MgSO₄	MW 120.37	Note* The MgSO₄ must be autoclaved separately.
1 ml	0.2% Thiamin (vitamin B1)

Final nutrient concentrations:

67.4 mM	Phosphate (PO₄^3-)
8.38 mM	Sulfate (SO₄^2-)
15.1 mM	Ammonium (NH₄)
5.1 mM	Sodium (Na⁺)
98.1 mM	Potassium (K⁺)
0.83 mM	Magnesium (MGgsup>2+)
1.7 mM	Citrate
139 µM	Glucose (in DM25)

NZY+ Broth

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

Add dH₂O to 950 ml. Adjust pH to 7.5 using NaOH (Requires ~350 µl of 10 N). Autoclave. Then add.

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

M9 Minimal

Make 5× M9 salts:

64 g	Sodium phosphate (dibasic, 7 hydrate) Na₂HPO₄•H₂O
15 g	Potassium phosphate (monobasic) KH₂PO₄
2.5 g	Sodium chloride, NaCl
5 g	Ammonium chloride NH₄Cl

Add dH₂O to 1 liter. Autoclave.

Combine the following ingredients (all pre-sterilized):

200 ml	5× M9 salts
2 ml	1M Magnesium sulfate MgSO₄
0.1 ml	1 M Calcium chloride CaCl₂
40 ml	10% (w/v) glucose (or other carbon source)

Add sterile dH₂O to 1 liter. Autoclave.

Solid Media

LB: Luria-Broth

Combine in a 2 L flask:

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.

Note: This is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

Added:

>
>

To make "soft" agar for phage studies, use only 7 g agar per liter.

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

Combine in a 2 L flask:

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:

6 g	Sugar (arabinose = TA, maltose = TM, lactose = TL)

Add dH₂O to 1.3 liter.

Autoclave sugar and media separately.

Combine and add:

1.5 ml

TTC (5%)

DM: Davis Minimal (MG, MA, MM, ML, ...)

10.5 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ H₂O
3 g	Potassium Phosphate (monobasic) H₂KPO₄
1.5 g	Ammonium Sulfate (NH₄)₂ SO₄
0.75 g	Sodium Citrate (tribasic, dihydrate) (Na₃Citrate 2•H₂O₄

Add dH₂O to 0.8 liter in a 2 L flask.

24 g / 10 g	Agar
1.5 ml	5% Antifoam

Add dH₂O to 0.5 liter in a 1 L flask.

6 g	Sugar (glucose = MG, arabinose = MA, maltose = MM, lactose = ML, ...

Add dH₂O to 0.2 liter in a 500 ml flask.

Autoclave. Important! Do not combine solutions before autoclaving. Agar and phosphate, phosphate and sugar, and agar and sugar react with each other at high temperatures to create compounds that inhibit bacterial cell growth.

Combine three flasks and add

1.5 ml	10% Magnesium Sulfate MgSO₄
1.5 ml	0.2% Thiamin (vitamin B1)

Total volume is 1.5 L, which makes 60-80 plates.

Revision 82011-03-08 - JeffreyBarrick

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

Media Recipes

Liquid Media

Changed:

<
<

Saline

>
>

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.

Changed:

<
<

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.

>
>

Included topic: ProtocolsRecipesSaline

Changed:

<
<

8.5 g

NaCl

>
>

Warning: Can't find topic Lab.ProtocolsRecipesLuriaBertani

Changed:

<
<

Add dH₂O to 1 liter. Autoclave.

>
>

Included topic: ProtocolsRecipesLuriaBertani

Deleted:

<
<

LB: Luria-Bertani

10 g	Tryptone
5 g	Yeast Extract
10 g	NaCl

Add dH₂O to 1 liter. Autoclave.

Note: This is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

DM: Davis Minimal

7 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ (H₂O)₃	MW 228.17
5 g	Potassium Phosphate (monobasic) H₂KPO₄	MW 136.07
1 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to 1 liter. Autoclave. Then add

1 ml	10% Magnesium Sulfate MgSO₄	MW 120.37	Note* The MgSO₄ must be autoclaved separately.
1 ml	0.2% Thiamin (vitamin B1)

Final nutrient concentrations:

67.4 mM	Phosphate (PO₄^3-)
8.38 mM	Sulfate (SO₄^2-)
15.1 mM	Ammonium (NH₄)
5.1 mM	Sodium (Na⁺)
98.1 mM	Potassium (K⁺)
0.83 mM	Magnesium (MGgsup>2+)
1.7 mM	Citrate
139 µM	Glucose (in DM25)

NZY+ Broth

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

Add dH₂O to 950 ml. Adjust pH to 7.5 using NaOH (Requires ~350 µl of 10 N). Autoclave. Then add.

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

M9 Minimal

Make 5× M9 salts:

64 g	Sodium phosphate (dibasic, 7 hydrate) Na₂HPO₄•H₂O
15 g	Potassium phosphate (monobasic) KH₂PO₄
2.5 g	Sodium chloride, NaCl
5 g	Ammonium chloride NH₄Cl

Add dH₂O to 1 liter. Autoclave.

Combine the following ingredients (all pre-sterilized):

200 ml	5× M9 salts
2 ml	1M Magnesium sulfate MgSO₄
0.1 ml	1 M Calcium chloride CaCl₂
40 ml	10% (w/v) glucose (or other carbon source)

Add sterile dH₂O to 1 liter. Autoclave.

Solid Media

LB: Luria-Broth

Combine in a 2 L flask:

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.

Note: This is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

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

Combine in a 2 L flask:

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:

6 g	Sugar (arabinose = TA, maltose = TM, lactose = TL)

Add dH₂O to 1.3 liter.

Autoclave sugar and media separately.

Combine and add:

1.5 ml

TTC (5%)

DM: Davis Minimal (MG, MA, MM, ML, ...)

10.5 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ H₂O
3 g	Potassium Phosphate (monobasic) H₂KPO₄
1.5 g	Ammonium Sulfate (NH₄)₂ SO₄
0.75 g	Sodium Citrate (tribasic, dihydrate) (Na₃Citrate 2•H₂O₄

Add dH₂O to 0.8 liter in a 2 L flask.

24 g / 10 g	Agar
1.5 ml	5% Antifoam

Add dH₂O to 0.5 liter in a 1 L flask.

6 g	Sugar (glucose = MG, arabinose = MA, maltose = MM, lactose = ML, ...

Add dH₂O to 0.2 liter in a 500 ml flask.

Autoclave. Important! Do not combine solutions before autoclaving. Agar and phosphate, phosphate and sugar, and agar and sugar react with each other at high temperatures to create compounds that inhibit bacterial cell growth.

Combine three flasks and add

1.5 ml	10% Magnesium Sulfate MgSO₄
1.5 ml	0.2% Thiamin (vitamin B1)

Total volume is 1.5 L, which makes 60-80 plates.

Revision 72011-03-07 - JeffreyBarrick

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

Media Recipes

Liquid Media

Saline

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.

8.5 g

NaCl

Add dH₂O to 1 liter. Autoclave.

LB: Luria-Bertani

10 g	Tryptone
5 g	Yeast Extract
10 g	NaCl

Add dH₂O to 1 liter. Autoclave.

Note: This is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

DM: Davis Minimal

7 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ (H₂O)₃	MW 228.17
5 g	Potassium Phosphate (monobasic) H₂KPO₄	MW 136.07
1 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to 1 liter. Autoclave. Then add

1 ml	10% Magnesium Sulfate MgSO₄	MW 120.37	Note* The MgSO₄ must be autoclaved separately.
1 ml	0.2% Thiamin (vitamin B1)

Final nutrient concentrations:

67.4 mM	Phosphate (PO₄^3-)
8.38 mM	Sulfate (SO₄^2-)
15.1 mM	Ammonium (NH₄)
5.1 mM	Sodium (Na⁺)
98.1 mM	Potassium (K⁺)
0.83 mM	Magnesium (MGgsup>2+)
1.7 mM	Citrate
139 µM	Glucose (in DM25)

NZY+ Broth

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

Add dH₂O to 950 ml. Adjust pH to 7.5 using NaOH (Requires ~350 µl of 10 N). Autoclave. Then add.

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

M9 Minimal

Make 5× M9 salts:

64 g	Sodium phosphate (dibasic, 7 hydrate) Na₂HPO₄•H₂O
15 g	Potassium phosphate (monobasic) KH₂PO₄
2.5 g	Sodium chloride, NaCl
5 g	Ammonium chloride NH₄Cl

Add dH₂O to 1 liter. Autoclave.

Combine the following ingredients (all pre-sterilized):

200 ml	5× M9 salts
2 ml	1M Magnesium sulfate MgSO₄
0.1 ml	1 M Calcium chloride CaCl₂
40 ml	10% (w/v) glucose (or other carbon source)

Add sterile dH₂O to 1 liter. Autoclave.

Solid Media

LB: Luria-Broth

Combine in a 2 L flask:

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.

Note: This is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

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

Combine in a 2 L flask:

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:

6 g	Sugar (arabinose = TA, maltose = TM, lactose = TL)

Add dH₂O to 1.3 liter.

Autoclave sugar and media separately.

Combine and add:

1.5 ml

TTC (5%)

DM: Davis Minimal (MG, MA, MM, ML, ...)

10.5 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ H₂O
3 g	Potassium Phosphate (monobasic) H₂KPO₄
1.5 g	Ammonium Sulfate (NH₄)₂ SO₄
0.75 g	Sodium Citrate (tribasic, dihydrate) (Na₃Citrate 2•H₂O₄

Add dH₂O to 0.8 liter in a 2 L flask.

24 g / 10 g	Agar
1.5 ml	5% Antifoam

Add dH₂O to 0.5 liter in a 1 L flask.

6 g	Sugar (glucose = MG, arabinose = MA, maltose = MM, lactose = ML, ...

Add dH₂O to 0.2 liter in a 500 ml flask.

Changed:

<
<

Autoclave. Important! Do not combine solutions before autoclaving. Agar and phosphate, phosphate and sugar, and agar and sugar react with each other at high temperatures to create compounds toxic to cells.

>
>

Autoclave. Important! Do not combine solutions before autoclaving. Agar and phosphate, phosphate and sugar, and agar and sugar react with each other at high temperatures to create compounds that inhibit bacterial cell growth.

Combine three flasks and add

1.5 ml	10% Magnesium Sulfate MgSO₄
1.5 ml	0.2% Thiamin (vitamin B1)

Total volume is 1.5 L, which makes 60-80 plates.

Revision 62011-02-28 - CraigBarnhart

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

Media Recipes

Liquid Media

Saline

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.

8.5 g

NaCl

Add dH₂O to 1 liter. Autoclave.

LB: Luria-Bertani

10 g	Tryptone
5 g	Yeast Extract
10 g	NaCl

Add dH₂O to 1 liter. Autoclave.

Note: This is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

DM: Davis Minimal

7 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ (H₂O)₃	MW 228.17
5 g	Potassium Phosphate (monobasic) H₂KPO₄	MW 136.07
1 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to 1 liter. Autoclave. Then add

Changed:

<
<

1 ml

10% Magnesium Sulfate MgSO₄

MW 120.37

>
>

1 ml

10% Magnesium Sulfate MgSO₄

MW 120.37

Note* The MgSO₄ must be autoclaved separately.

1 ml

0.2% Thiamin (vitamin B1)

Final nutrient concentrations:

67.4 mM	Phosphate (PO₄^3-)
8.38 mM	Sulfate (SO₄^2-)
15.1 mM	Ammonium (NH₄)
5.1 mM	Sodium (Na⁺)
98.1 mM	Potassium (K⁺)
0.83 mM	Magnesium (MGgsup>2+)
1.7 mM	Citrate
139 µM	Glucose (in DM25)

NZY+ Broth

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

Add dH₂O to 950 ml. Adjust pH to 7.5 using NaOH (Requires ~350 µl of 10 N). Autoclave. Then add.

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

M9 Minimal

Make 5× M9 salts:

64 g	Sodium phosphate (dibasic, 7 hydrate) Na₂HPO₄•H₂O
15 g	Potassium phosphate (monobasic) KH₂PO₄
2.5 g	Sodium chloride, NaCl
5 g	Ammonium chloride NH₄Cl

Add dH₂O to 1 liter. Autoclave.

Combine the following ingredients (all pre-sterilized):

200 ml	5× M9 salts
2 ml	1M Magnesium sulfate MgSO₄
0.1 ml	1 M Calcium chloride CaCl₂
40 ml	10% (w/v) glucose (or other carbon source)

Add sterile dH₂O to 1 liter. Autoclave.

Solid Media

LB: Luria-Broth

Combine in a 2 L flask:

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.

Note: This is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

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

Combine in a 2 L flask:

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:

6 g	Sugar (arabinose = TA, maltose = TM, lactose = TL)

Add dH₂O to 1.3 liter.

Autoclave sugar and media separately.

Combine and add:

1.5 ml

TTC (5%)

DM: Davis Minimal (MG, MA, MM, ML, ...)

10.5 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ H₂O
3 g	Potassium Phosphate (monobasic) H₂KPO₄
1.5 g	Ammonium Sulfate (NH₄)₂ SO₄
0.75 g	Sodium Citrate (tribasic, dihydrate) (Na₃Citrate 2•H₂O₄

Add dH₂O to 0.8 liter in a 2 L flask.

24 g / 10 g	Agar
1.5 ml	5% Antifoam

Add dH₂O to 0.5 liter in a 1 L flask.

6 g	Sugar (glucose = MG, arabinose = MA, maltose = MM, lactose = ML, ...

Add dH₂O to 0.2 liter in a 500 ml flask.

Autoclave. Important! Do not combine solutions before autoclaving. Agar and phosphate, phosphate and sugar, and agar and sugar react with each other at high temperatures to create compounds toxic to cells.

Combine three flasks and add

1.5 ml	10% Magnesium Sulfate MgSO₄
1.5 ml	0.2% Thiamin (vitamin B1)

Total volume is 1.5 L, which makes 60-80 plates.

Revision 52011-02-27 - JeffreyBarrick

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

Media Recipes

Liquid Media

Added:

>
>

Saline

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.

8.5 g

NaCl

Add dH₂O to 1 liter. Autoclave.

LB: Luria-Bertani

10 g	Tryptone
5 g	Yeast Extract
10 g	NaCl

Add dH₂O to 1 liter. Autoclave.

Note: This is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

DM: Davis Minimal

7 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ (H₂O)₃	MW 228.17
5 g	Potassium Phosphate (monobasic) H₂KPO₄	MW 136.07
1 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to 1 liter. Autoclave. Then add

1 ml	10% Magnesium Sulfate MgSO₄	MW 120.37
1 ml	0.2% Thiamin (vitamin B1)

Final nutrient concentrations:

67.4 mM	Phosphate (PO₄^3-)
8.38 mM	Sulfate (SO₄^2-)
15.1 mM	Ammonium (NH₄)
5.1 mM	Sodium (Na⁺)
98.1 mM	Potassium (K⁺)
0.83 mM	Magnesium (MGgsup>2+)
1.7 mM	Citrate
139 µM	Glucose (in DM25)

NZY+ Broth

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

Add dH₂O to 950 ml. Adjust pH to 7.5 using NaOH (Requires ~350 µl of 10 N). Autoclave. Then add.

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

M9 Minimal

Make 5× M9 salts:

64 g	Sodium phosphate (dibasic, 7 hydrate) Na₂HPO₄•H₂O
15 g	Potassium phosphate (monobasic) KH₂PO₄
2.5 g	Sodium chloride, NaCl
5 g	Ammonium chloride NH₄Cl

Add dH₂O to 1 liter. Autoclave.

Combine the following ingredients (all pre-sterilized):

200 ml	5× M9 salts
2 ml	1M Magnesium sulfate MgSO₄
0.1 ml	1 M Calcium chloride CaCl₂
40 ml	10% (w/v) glucose (or other carbon source)

Add sterile dH₂O to 1 liter. Autoclave.

Solid Media

LB: Luria-Broth

Combine in a 2 L flask:

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.

Note: This is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

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

Combine in a 2 L flask:

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:

6 g	Sugar (arabinose = TA, maltose = TM, lactose = TL)

Add dH₂O to 1.3 liter.

Autoclave sugar and media separately.

Combine and add:

1.5 ml

TTC (5%)

DM: Davis Minimal (MG, MA, MM, ML, ...)

10.5 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ H₂O
3 g	Potassium Phosphate (monobasic) H₂KPO₄
1.5 g	Ammonium Sulfate (NH₄)₂ SO₄
0.75 g	Sodium Citrate (tribasic, dihydrate) (Na₃Citrate 2•H₂O₄

Add dH₂O to 0.8 liter in a 2 L flask.

24 g / 10 g	Agar
1.5 ml	5% Antifoam

Add dH₂O to 0.5 liter in a 1 L flask.

6 g	Sugar (glucose = MG, arabinose = MA, maltose = MM, lactose = ML, ...

Add dH₂O to 0.2 liter in a 500 ml flask.

Autoclave. Important! Do not combine solutions before autoclaving. Agar and phosphate, phosphate and sugar, and agar and sugar react with each other at high temperatures to create compounds toxic to cells.

Combine three flasks and add

1.5 ml	10% Magnesium Sulfate MgSO₄
1.5 ml	0.2% Thiamin (vitamin B1)

Total volume is 1.5 L, which makes 60-80 plates.

Revision 42010-07-23 - JeffreyBarrick

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

Media Recipes

Liquid Media

LB: Luria-Bertani

10 g	Tryptone
5 g	Yeast Extract
10 g	NaCl

Add dH₂O to 1 liter. Autoclave.

Note: This is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

DM: Davis Minimal

7 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ (H₂O)₃	MW 228.17
5 g	Potassium Phosphate (monobasic) H₂KPO₄	MW 136.07
1 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to 1 liter. Autoclave. Then add

1 ml	10% Magnesium Sulfate MgSO₄	MW 120.37
1 ml	0.2% Thiamin (vitamin B1)

Final nutrient concentrations:

67.4 mM	Phosphate (PO₄^3-)
8.38 mM	Sulfate (SO₄^2-)
15.1 mM	Ammonium (NH₄)
5.1 mM	Sodium (Na⁺)
98.1 mM	Potassium (K⁺)
0.83 mM	Magnesium (MGgsup>2+)
1.7 mM	Citrate
139 µM	Glucose (in DM25)

NZY+ Broth

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

Add dH₂O to 950 ml. Adjust pH to 7.5 using NaOH (Requires ~350 µl of 10 N). Autoclave. Then add.

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

M9 Minimal

Changed:

<
<

Make M9 salts:

>
>

Make 5× M9 salts:

64 g	Sodium phosphate (dibasic, 7 hydrate) Na₂HPO₄•H₂O
15 g	Potassium phosphate (monobasic) KH₂PO₄

Changed:

<
<

2.5 g

Sodiu chloride, NaCl

>
>

2.5 g

Sodium chloride, NaCl

5 g	Ammonium chloride NH₄Cl

Add dH₂O to 1 liter. Autoclave.

Combine the following ingredients (all pre-sterilized):

Changed:

<
<

200 ml	M9 salts
2 ml	Magnesium sulfate MgSO₄
0.1 ml	1 M CaCl₂

>
>

200 ml	5× M9 salts
2 ml	1M Magnesium sulfate MgSO₄
0.1 ml	1 M Calcium chloride CaCl₂

40 ml

10% (w/v) glucose (or other carbon source)

Add sterile dH₂O to 1 liter. Autoclave.

Solid Media

LB: Luria-Broth

Combine in a 2 L flask:

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.

Note: This is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

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

Combine in a 2 L flask:

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:

6 g	Sugar (arabinose = TA, maltose = TM, lactose = TL)

Add dH₂O to 1.3 liter.

Autoclave sugar and media separately.

Combine and add:

1.5 ml

TTC (5%)

DM: Davis Minimal (MG, MA, MM, ML, ...)

10.5 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ H₂O
3 g	Potassium Phosphate (monobasic) H₂KPO₄
1.5 g	Ammonium Sulfate (NH₄)₂ SO₄
0.75 g	Sodium Citrate (tribasic, dihydrate) (Na₃Citrate 2•H₂O₄

Add dH₂O to 0.8 liter in a 2 L flask.

24 g / 10 g	Agar
1.5 ml	5% Antifoam

Add dH₂O to 0.5 liter in a 1 L flask.

6 g	Sugar (glucose = MG, arabinose = MA, maltose = MM, lactose = ML, ...

Add dH₂O to 0.2 liter in a 500 ml flask.

Autoclave. Important! Do not combine solutions before autoclaving. Agar and phosphate, phosphate and sugar, and agar and sugar react with each other at high temperatures to create compounds toxic to cells.

Combine three flasks and add

1.5 ml	10% Magnesium Sulfate MgSO₄
1.5 ml	0.2% Thiamin (vitamin B1)

Total volume is 1.5 L, which makes 60-80 plates.

Revision 32010-07-22 - JeffreyBarrick

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

Media Recipes

Liquid Media

LB: Luria-Bertani

10 g	Tryptone
5 g	Yeast Extract
10 g	NaCl

Add dH₂O to 1 liter. Autoclave.

Note: This is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

DM: Davis Minimal

7 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ (H₂O)₃	MW 228.17
5 g	Potassium Phosphate (monobasic) H₂KPO₄	MW 136.07
1 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to 1 liter. Autoclave. Then add

1 ml	10% Magnesium Sulfate MgSO₄	MW 120.37
1 ml	0.2% Thiamin (vitamin B1)

Final nutrient concentrations:

67.4 mM	Phosphate (PO₄^3-)
8.38 mM	Sulfate (SO₄^2-)
15.1 mM	Ammonium (NH₄)
5.1 mM	Sodium (Na⁺)
98.1 mM	Potassium (K⁺)
0.83 mM	Magnesium (MGgsup>2+)
1.7 mM	Citrate
139 µM	Glucose (in DM25)

NZY+ Broth

Added:

>
>

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

Add dH₂O to 950 ml. Adjust pH to 7.5 using NaOH (Requires ~350 µl of 10 N). Autoclave. Then add.

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

Added:

>
>

M9 Minimal

Make M9 salts:

64 g	Sodium phosphate (dibasic, 7 hydrate) Na₂HPO₄•H₂O
15 g	Potassium phosphate (monobasic) KH₂PO₄
2.5 g	Sodiu chloride, NaCl
5 g	Ammonium chloride NH₄Cl

Add dH₂O to 1 liter. Autoclave.

Combine the following ingredients (all pre-sterilized):

200 ml	M9 salts
2 ml	Magnesium sulfate MgSO₄
0.1 ml	1 M CaCl₂
40 ml	10% (w/v) glucose (or other carbon source)

Add sterile dH₂O to 1 liter. Autoclave.

Solid Media

LB: Luria-Broth

Combine in a 2 L flask:

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.

Note: This is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

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

Combine in a 2 L flask:

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:

6 g	Sugar (arabinose = TA, maltose = TM, lactose = TL)

Add dH₂O to 1.3 liter.

Autoclave sugar and media separately.

Combine and add:

1.5 ml

TTC (5%)

DM: Davis Minimal (MG, MA, MM, ML, ...)

10.5 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ H₂O
3 g	Potassium Phosphate (monobasic) H₂KPO₄
1.5 g	Ammonium Sulfate (NH₄)₂ SO₄
0.75 g	Sodium Citrate (tribasic, dihydrate) (Na₃Citrate 2•H₂O₄

Add dH₂O to 0.8 liter in a 2 L flask.

24 g / 10 g	Agar
1.5 ml	5% Antifoam

Add dH₂O to 0.5 liter in a 1 L flask.

6 g	Sugar (glucose = MG, arabinose = MA, maltose = MM, lactose = ML, ...

Add dH₂O to 0.2 liter in a 500 ml flask.

Autoclave. Important! Do not combine solutions before autoclaving. Agar and phosphate, phosphate and sugar, and agar and sugar react with each other at high temperatures to create compounds toxic to cells.

Combine three flasks and add

1.5 ml	10% Magnesium Sulfate MgSO₄
1.5 ml	0.2% Thiamin (vitamin B1)

Total volume is 1.5 L, which makes 60-80 plates.

Revision 22010-05-12 - JeffreyBarrick

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

Changed:

<
<

Media Recipes

>
>

Added:

>
>

Media Recipes

Changed:

<
<

Davis Minimal (DM) Media

>
>

Liquid Media

Changed:

<
<

Luria Broth (LB)

>
>

LB: Luria-Bertani

Changed:

<
<

Luria Broth (LB) Agar

>
>

10 g

Tryptone

Added:

>
>

5 g	Yeast Extract
10 g	NaCl

Changed:

<
<

Minimal Glucose (MG) // Minimal Arabinose (MA) Agar

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Add dH₂O to 1 liter. Autoclave.

Changed:

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Tetrazolium Arabinose (TA) Agar

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Note: This is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

Changed:

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Other sugars such as maltose and lactose can also be used.

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DM: Davis Minimal

Added:

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7 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ (H₂O)₃	MW 228.17
5 g	Potassium Phosphate (monobasic) H₂KPO₄	MW 136.07
1 g	Ammonium Sulfate (NH₄)₂ SO₄	MW 132.08
0.5 g	Sodium Citrate (trisodium, dihydrate) Na₃C₆H₅O₇ (H₂O)₂	MW 294.10

Add dH₂O to 1 liter. Autoclave. Then add

1 ml	10% Magnesium Sulfate MgSO₄	MW 120.37
1 ml	0.2% Thiamin (vitamin B1)

Final nutrient concentrations:

67.4 mM	Phosphate (PO₄^3-)
8.38 mM	Sulfate (SO₄^2-)
15.1 mM	Ammonium (NH₄)
5.1 mM	Sodium (Na⁺)
98.1 mM	Potassium (K⁺)
0.83 mM	Magnesium (MGgsup>2+)
1.7 mM	Citrate
139 µM	Glucose (in DM25)

NZY+ Broth

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

Add dH₂O to 950 ml. Adjust pH to 7.5 using NaOH (Requires ~350 µl of 10 N). Autoclave. Then add.

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

Solid Media

LB: Luria-Broth

Combine in a 2 L flask:

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.

Note: This is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

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

Combine in a 2 L flask:

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:

6 g	Sugar (arabinose = TA, maltose = TM, lactose = TL)

Add dH₂O to 1.3 liter.

Autoclave sugar and media separately.

Combine and add:

1.5 ml

TTC (5%)

DM: Davis Minimal (MG, MA, MM, ML, ...)

10.5 g	Potassium Phosphate (dibasic, trihydrate) HK₂PO₄ H₂O
3 g	Potassium Phosphate (monobasic) H₂KPO₄
1.5 g	Ammonium Sulfate (NH₄)₂ SO₄
0.75 g	Sodium Citrate (tribasic, dihydrate) (Na₃Citrate 2•H₂O₄

Add dH₂O to 0.8 liter in a 2 L flask.

24 g / 10 g	Agar
1.5 ml	5% Antifoam

Add dH₂O to 0.5 liter in a 1 L flask.

6 g	Sugar (glucose = MG, arabinose = MA, maltose = MM, lactose = ML, ...

Add dH₂O to 0.2 liter in a 500 ml flask.

Autoclave. Important! Do not combine solutions before autoclaving. Agar and phosphate, phosphate and sugar, and agar and sugar react with each other at high temperatures to create compounds toxic to cells.

Combine three flasks and add

1.5 ml	10% Magnesium Sulfate MgSO₄
1.5 ml	0.2% Thiamin (vitamin B1)

Total volume is 1.5 L, which makes 60-80 plates.

Revision 12010-05-11 - JeffreyBarrick

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

Difference: ProtocolsMediaRecipes (1 vs. 48)

Revision 482025-06-12 - AlexaMorton

Media Recipes

Liquid Media

Sterile Saline

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

DM: Davis-Mingioli

M9 Minimal Medium

ABMS: Acinetobacter Minimal Succinate

ABMS Component Recipes

R2A

RCV Medium

SOB/SOC: Super Optimal Broth

For SOC (=SOB+glucose)

Containers for SOB/SOC

TGY Medium

DR: Defined minimal media for D. radiodurans

YPS Medium

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

YPD Medium

Czapek Broth (CB)

Solid Media

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

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

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

MC: Minimal Citrate

M9 Minimal Media Plates

PA: Lac Papillation Agar

TGY Medium

MOB: Mobility Media

Blood - Heart Infusion Agar

Stab Agar

Revision 472024-10-16 - MeghnaVergis

Media Recipes

Liquid Media

Sterile Saline

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

DM: Davis-Mingioli

M9 Minimal Medium

ABMS: Acinetobacter Minimal Succinate

ABMS Component Recipes

R2A

RCV Medium

SOB/SOC: Super Optimal Broth

For SOC (=SOB+glucose)

Containers for SOB/SOC

TGY Medium

DR: Defined minimal media for D. radiodurans

YPS Medium

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

YPD Medium

Czapek Broth (CB)

Solid Media

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

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

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

MC: Minimal Citrate

M9 Minimal Media Plates

PA: Lac Papillation Agar

TGY Medium

MOB: Mobility Media

Blood - Heart Infusion Agar

Stab Agar

Revision 462024-07-09 - MeghnaVergis

Media Recipes

Liquid Media

Sterile Saline

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

DM: Davis-Mingioli

M9 Minimal Medium

ABMS: Acinetobacter Minimal Succinate

ABMS Component Recipes

R2A

RCV Medium

SOB/SOC: Super Optimal Broth

For SOC (=SOB+glucose)

Containers for SOB/SOC

TGY Medium

DR: Defined minimal media for D. radiodurans

YPS Medium