M9 medium composition

M9 is one of the standard minimal medium for bacterial fermentation. Here is a procedure to prepare this medium with sucrose as a sole carbon source. Alternatively, other sugars such as glucose, lactose, or maltose can be used.

M9 medium with sucrose as C-source

Prepare stock solution

  1. Salts 10x-stock solution (solution A)

Component

Concentration (mM)

Concentration [g/l]

Na2HPO4

422.5

60

KH2PO4

220.6

30

NaCl

85.5

5

NH4Cl

46.7

10

Procedure:

Make one-liter 10x stock solution and sterile filtered using Millipore Sterivex membrane filter

  1. Sucrose stock solution (solution B)

Component

Concentration (mM)

Concentration [g/l]

sucrose

730.4

Procedure:

Make 500-ml stock solution and sterile filtered using Millipore Sterivex membrane filter

  1. MgSO4.7H2O 1 M, filter sterile
  2. CaCl2 1 M, filter sterile
  3. Thiamine-HCl 10 g/l, filter sterile
  4. Distilled water, autoclaved

Procedure for preparing one-liter M9 medium with 0.2 % w/v sucrose

  1. Prepare 890 ml sterile water in one-liter sterile bottle
  2. Add 100 ml of CaCl2 1 M, mixed well to avoid precipitation during the sulphate salt addition
  3. Add 100 ml of Thiamine-HCl 10 g/l
  4. Add 1 ml MgSO4.7H2O 1 M
  5. Add 100 ml M9 salts (10x)
  6. Add 8 ml sucrose 250 g/l (25 % w/v)

To avoid precipitation, see https://yalun.wordpress.com/2008/12/20/precipitation-during-m9-medium-preparation/

Final concentration:

Component

Concentration (mM)

Concentration

[g/l][mg/l]

Na2HPO4

42.25

6

KH2PO4

22.06

3

NaCl

8.55

0.5

NH4Cl

18.7

1

Sucrose

5.84

2

MgSO4.7H2O

1

246

CaCl2.2H2O

0.1

14.7

Thiamine-HCl

0.003

1

4 Responses

  1. “Procedure for preparing one-liter M9 medium with 0.2 % w/v sucrose
    Prepare 890 ml sterile water in one-liter sterile bottle
    Add 100 ml of CaCl2 1 M, mixed well to avoid precipitation during the sulphate salt addition
    Add 100 ml of Thiamine-HCl 10 g/l
    Add 1 ml MgSO4.7H2O 1 M
    Add 100 ml M9 salts (10x)
    Add 8 ml sucrose 250 g/l (25 % w/v)….”

    The volume is lager than 1 liter – there must be some mistake ?!!

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