M9 medium


Written 2022-09-23

M9 medium (otherwise known from here as M9) is a chemically defined medium used to grow Escherichia coli. I am primarily interested in M9 media because it is chemically defined.

M9 recipe I use

M9 is a simple media with several salts, glucose, and trace elements. Nitrogen source is ammonium chloride, carbon source is glucose, and phosphate source is both disodium phosphate + monopotassium phosphate. Confusingly, there are several different recipes for M9, so I will try to explain the reasoning behind each ingredient.

TODO: add my full recipe in glorious text.

History of M9 medium

In 1869, the first true chemically defined medium was developed for Aspergillus niger by Raulin, a student of Pasteur, in his paper "Études chimiques sur la végétation" (written in French).

Études chimiques sur la végétation

The first mention of M-9 medium (which will be referred to as M9) was in "MUTATIONS IN BACTERIA INDUCED BY RADIATIONS" by M. Demerec and R. Latarjet, published in 1946. In it, they name M9 as "the laboratory name for an ammonium chloride-glucose-phosphate buffer medium developed by E. H. Anderson (1)" in his paper "Growth Requirements of Virus-Resistant Mutants of Escherichia Coli Strain “B”".


Growth Requirements of Virus-Resistant Mutants of Escherichia Coli Strain “B”

Trace elements

Most M9 medium compositions do not include trace minerals, such as iron. This wasn't really noticed back in the days of the original experiments - most distilled water still came from metal pipes, and these metal pipes would release a tiny amount of metal, not enough for easy detectability, but enough for growth. Modern labs with distilled water systems may need to add these extra trace metals to support robust growth.

E.coli needs iron to grow quickly.

Glucose vs Glycerol

Some M9 recipes use glucose and some use glycerol as the carbon/energy source. Both are roughly equivalent. Glycerol may be easier in low-resourced labs since its natural syrup state should be fairly resistant to contamination (citation needed, just theory), similar to honey. A very high percentage glucose stock would have to be made to be roughly equivalent - at least above 60%.

Glucose and glycerol support similar growth rates.

Yeasts can grow in 60% sugar.

Other carbon sources can also be used, like pyruvate, at a lower efficiency. It is especially important to not autoclave glucose in the presence of other amino acids, since this will cause the maillard reaction to chemically change both substances. Thankfully, M9 does not include any amino acids, but the glucose can still caramelize.

MgSO4 and CaCl2

M9 recipes require that MgSO4 and CaCl2 get added separately. This is due to the fact that CaSO4 will be produced when CaCl2 reacts with MgSO4 in mixture. CaS04 has a much lower solubility in water (0.26 g/100ml at 25c) than either CaCl2 (74.5 g/100 mL at 20c) or MgSO4 (74.5 g/100 mL at 20c). While this is a problem when the mixtures are concentrated, it is not usually a problem with the final stock of M9.

MgSO4 and CaCl2 are added as separate solutions to prevent precipitation problems


Many commonly used Escherichia coli strains are thiamine (Vitamin B1) auxotrophs. Therefore, it is important to add a small amount to M9 to support their growth. 1mg per L is enough to support growth.

Thiamin stock solution should be added


Mysteriously, biotin is sometimes added to M9 media as a part of the trace vitamins. However, Escherichia coli is able to synthesize biotin itself.

E.coli can synthesize biotin

Notes on common strains

Escherichia coli DH5α

DH5α grows slowly on M9 due to a purB mutation

Due to this, if you're working with Escherichia coli, it is recommended you use a strain like NEB Turbos instead.

Bacillus subtilis 168

Usage of M9 with Bacillus subtilis 168.

Apparently, trp- strains like Bacillus subtilis 168 grow quite poorly in M9, even with trp added.


Thank you to Sebastian Cocioba for the history of M9. It's nearly impossible to find online.