M9 medium


M9 is a minimal, low osmolarity media for E. Coli, resulting in slower growth rate of these cells.


5X Stock

  • Na2HPO4
  • KH2PO4
  • NH4Cl
  • NaCl
  • CaCl2 (optional)

Additional Components for 1X Working Solution

  • MgSO4.7H2O
  • Carbon source (sugar or glycerol)
  • 0.5% vitamin B1 (thiamine)
  • 20% Casamino Acids or L amino acids to 40 ug/ml or DL amino acids to 80 ug/ml
  • Antibiotic


Preparing the 5X stock

Add the following reagents to a 2-liter flask:

  • 30 g Na2HPO4
  • 15 g KH2PO4
  • 5 g NH4Cl
  • 2.5 g NaCl
  • 15 mg CaCl2 (optional)
  • 1 liter of high quality distilled water

Once the ingredients are added, heat with stirring until the components are completely dissolved. Pour the solution into smaller bottles with loosened caps and the autoclave at 15 lb/in2 for 15 min. If you wish to add add  antibiotics or nutritional supplements, do this only after the autoclave cycle is complete, as the high temperature may destroy these components. Wait until the the bottle is less than 50°C (it should be warm to touch), and then add the components.  After the bottles cool to below 40°C, the caps can be tightened and the concentrated medium stored indefinitely at room temperature.

Preparing the 1X Working Solution

To make 1X working solution, the 5X media should be diluted to 1× with high quality sterile distilled water.

Add the following sterile solutions

  • 1 ml 1 M MgSO4.7H2O
  • 10 ml 20% carbon source (sugar or glycerol)

Typically, several additional components are also added to make a complete medium.

  • 0.1 ml 0.5% vitamin B1 (thiamine)
  • 5 ml 20% Casamino Acids or L amino acids to 40 ug/ml or DL amino acids to 80 ug/ml
  • Antibiotic


Formulation sourced from an article by Karen Elbing and Roger Brent. Media Preparation and Bacteriological Tools. Current Protocols in Molecular Biology (2002) 1.1.1-1.1.7.

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