M9 Minimal Salt Media, NREL Formulation

This formulation of minimal salt growth media was originally described by Linger et al (10.1073/pnas.1410657111) for the cultivation of Pseudomonas putida KT2440. We have made slight modifications since adopting it in our lab. Growth is a bit more robust than the conventional M9 Maniatis recipie used for E. coli that is commercially available from Sigma as a 5X powder, and much better than de Bont minimal media (DOI: 10.1128/aem.55.11.2850-2855.1989).

New trainees: a common mistake that we have seen is to simply dilute 10X M9 salts and use as a 1X solution, sometimes even without adding glucose. There are a few more additives which precipitate out if stored in the 10x form and must be added separately.

10X M9 Media Stock

Per 1L Media

(NH4)2SO4 20 g
Na2HPO4 68 g
KH2PO4 30 g
NaCl 5 g

Variant: NH4Cl. Substitute 53.5 g NH4Cl in place of (NH4)2SO4. These are the working nitrogen concentrations we use.

1x M9 Minimal Salt Media

Per 1L Media

10X M9 stock 100 mL
1M MgSO4 2 mL
1M CaCl2 100 µL
Carbon
(usually stock
20% glucose) 50 mL
trace elements 500 µL
ddH20 to 1L (usually around 850mLs)

Don't filter sterilize the trace elements. If you are not using sterile water and need to filter sterilize, mix all the components together except the trace elements to filter sterilize. Then afterwards, add the trace elements to the 1X M9 media.

We use the Teknova Trace elements solution:

10mM Zinc Sulfate

10mM Manganese Chloride

2mM Cobalt Chloride

2mM Cupric Chloride

2mM Sodium Molybdate

20mM Calcium Chloride

50mM Ferric Chloride

2mM Boric Acid

2mM Nickel Chloride

2mM Sodium Selenite