Preparation of M9 Minimal Medium


M9 minimal medium is a chemically-defined minimal growth medium, used to cultivate Escherichia coli. It is prepared by supplementing M9 Minimal Salt solution with an appropriate carbon and energy source, and some trace elements.

The following section describes a procedure to prepare 1000 ml of M9 minimal medium.


Reagents and solutions
5x M9 Minimal Salt solution
20% Glucose (sugar or glycerol)
1 M Magnesium sulfate (MgSO4)
1M CaCl2 
Deionized water

0.5% vitamin B1 (thiamine)
20% Casamino Acids

Equipment and disposables
Measuring cylinder
Conical flask / Beaker
Magnetic stirrer

Composition of M9 Minimal medium
1x M9 Minimal salts
0.2% Glucose
2 mM MgSO4
0.1 mM CaCl2

Preparation of 1000 ml of M9 Minimal medium


Step 1: To prepare 1000 ml of M9 Minimal medium, take 700 ml water in a 2-liter beaker. Add 200 ml 5x Minimal salts, 10 ml of 20% Glucose, 1 ml of 1M MgSO4, and 0.1 ml of 1M CaCl2. Mix.

Note: if required, 0.1 ml 0.5% vitamin B1 (thiamine), 5 ml 20% Casamino Acids, and antibiotics can be added.

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

Step 3: Sterilize the solution by filtering through a filter unit containing a membrane with pore size 0.22 or less under aseptic conditions.

You don’t need to filter-sterilize the solution if you have used sterile reagents and glasswares and handled them aseptically.

Store the solution in fridge (2 – 8°C)

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