Preparation of 5x A Minimal Medium Without Supplements


A Minimal Medium is a chemically-defined growth medium. It  can be used to culture Escherichia coli. It is prepared as a concentrated medium without supplement which is diluted to a working solution and supplemented with appropriate carbon and energy source, and some trace elements. 

A 1000 ml of 5x concentrated medium is prepared by dissolving 10 g Ammonium Sulfate {(NH4)2SO4}, 68 g Potassium Phosphate Monobasic (KH2PO4), and 2.5 mg Sodium citrate dihydrate in water to a final volume of 1000 ml. Autoclaved 5x concentrated medium is quite stable and can be stored at room temperature for years. 

Working solution is prepared by diluting and supplementing the concentrated medium with MgSO4 and a carbon source e.g., sugar or glycerol. Depending on the requirements, vitamin B1, Casamino Acids, and antibiotics can also be added in the medium.


Reagents and solutions
Ammonium Sulfate {(NH4)2SO4, Molecular Weight:132.14)}
Potassium dihydrogen phosphate (KH2PO4, Molecular Weight: 136.09)
Potassium phosphate dibasic (K2HPO4, Molecular Weight: 174.18)
Sodium citrate dihydrate {HOC(COONa)(CH2COONa)2.2H2O; Molecular Weight: 294.10}
Deionized water

Equipment and disposables
Measuring cylinder
Conical flask / Beaker
Magnetic stirrer

Composition of 5x A Medium (without supplements)
37.8 mM (NH4)2SO4
165 mM KH2PO4
301 mM K2HPO4
8.5 mM Sodium citrate dihydrate

Preparation of 1000 ml of 5x A Medium (without supplements)


Step 1: To prepare 1000 ml of 5x A Minimal medium (without supplement), weigh out 5 g (NH4)2SO4, 22.5 g KH2PO4, 52.5 g K2HPO4 and 2.5 g sodium citrate dihydrate in a 2-liter conical flask/beaker. Add 800 ml deionized/Milli-Q water. Stir until all ingredients are completely dissolved.

◊ You can heat the solution to dissolve all solutes.

◊ One can use manual shaking using a glass pipette to mix all ingredients. Mixing using a Magnetic stirrer is optional. The magnetic stirrer makes the dissolving process easy and convenient.

◊ Do not dissolve in 1000 ml of deionized / Milli-Q water. It is always a good practice to dissolve all ingredients first and then adjust the volume to the desired volume with the solvent. In most cases, solution volume increases when a large amount of solute dissolves in the solvent.

◊ Make sure to dissolve any residual powder sticking to the glass.

Step 2: Adjust the volume to 1000 ml with deionized water. Mix it again.

Step 3: Transfer the medium to an autoclavable bottle. Sterilize the solution by autoclaving (20 minutes at 15 lb/ (psi) from 121-124°C on liquid cycle).

The solution can be stored at room temperature or in a cold room.

Preparation of A Minimal growth medium

Follow the table to prepare 5x A Minimal medium (without supplements) of various volumes
Ingredients/Volume100 ml250 ml500 ml1000 ml
(NH4)2SO40.5 g1.25 g2.5 g5 g 
KH2PO42.25 g5.625 g11.25 g22.5 g
K2HPO45.25 g13.125 g26.25 g52.5 g
Sodium citrate dihydrate0.250.6251.252.5
WaterTo 100 mlTo 250 mlTo 500 mlTo 1000 ml

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