Preparation of 5x M9 Minimal Salts

M9 Minimal Salts serve as a base salt solution to prepare M9 Minimal Medium. M9 Minimal salts solution is quite stable at room temperature and has a long shelf life. When supplemented with an appropriate carbon and energy source, and some trace elements, it is called M9 minimal medium. M9 minimal medium is a chemically-defined minimal growth medium, used to cultivate Escherichia coli

Usually, the M9 Minimal Salt solution is prepared as a 5x concentrated solution which must be diluted to 1x final concentration to prepare M9 Minimal Medium. The 5x M9 Minimal Salt solution contains 210 mM Na2HPO4, 110 mM KH2PO4, 42.8 mM NaCl, and 93 mM NH4Cl. A 1000 ml 5x  M9 Minimal Salts solution is prepared by dissolving 30 g Na2HPO4 (anhydrous), 15 g KH2PO4, 2.5g NaCl, and 5g NH4Cl in water to a final volume of 1000 ml.


Reagents and solutions
Disodium hydrogen phosphate, anhydrous (Na2HPO4, Mol wt: 141.96)
Potassium dihydrogen phosphate (KH2PO4, Mol wt: 136.09)
Sodium chloride (NaCl, Mol wt: 58.44)
Ammonium chloride (NH4Cl, Mol wt: 53.49)
Deionized water

Equipment and disposables
Measuring cylinder
Conical flask / Beaker
Magnetic stirrer

Composition of 5x M9 Minimal Salts
210 mM Na2HPO4 *
110 mM KH2PO4
42.8 mM NaCl
93 mM NH4Cl

* You can find some variations in the concentration of Na2HPO4 (varies from 210 – 240 mM) from different sources.

Preparation of 1000 ml of 5x M9 Minimal Salts


Step 1: To prepare 1000 ml of 5x M9 Minimal salts, weigh out 30 g Na2HPO4 (anhydrous), 15 g KH2PO4, 2.5g NaCl, and 5g NH4Cl in a 2-liter conical flask/beaker. Add 800 ml deionized/Milli-Q water. Stir until all ingredients are completely dissolved.

If you don’t have anhydrous Na2HPO4 and have a hydrated hydrogen phosphate (Na2HPO4) in your lab:

  • Add 37.4 g of Na2HPO4.2H2O (Mol Wt: 177.99)
  • Add 56.3 g of  Na2HPO4.7H2O (Mol. Wt: 268.07)

◊ You can heat the solution to accelerate the dissolving process.

◊ 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 water. It is always a good practice to dissolve all media 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 M9 Minimal growth medium

Follow the table to prepare 5x M9 Minimal Salts of various volumes.
Ingredients / Volume100 ml250 ml500 ml1000 ml
Na2HPO43 g7.5 g15 g30 g 
KH2PO41.5 g3.75 g7.5 g15 g
NaCl0.25 g0.625 g1.25 g2.5 g
NH4Cl0.5 g1.25 g2.5 g5 g

Was this post helpful?

Author: admin

Leave a Reply

Your email address will not be published. Required fields are marked *