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.

REQUIREMENTS

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.

Objective
Preparation of 1000 ml of 5x M9 Minimal Salts

PROCEDURE

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.

Note:
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)

Tips:
◊ 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.

Precautions:
◊ 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/sq.in. (psi) from 121-124°C on liquid cycle).

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

Applications
Preparation of M9 Minimal growth medium