M9 minimal medium

This medium is used to grow B. subtilis in the presence of glucose or other variable carbon sources. The medium does contain ammonium and is therefore not suited to change the nitrogen source.

The basal solutions

• 5x M9 stock solution, per liter
  • 42.5 g Na2HPO4.2H2O (Merck)
  • 15 g KH2PO4 (Merck)
  • 5.0 g NH4Cl (Merck)
  • 2.5 g NaCl (Merck)

• • Adjust to pH 7.0 using 4M NaOH. Solution is autoclavable at 121oC for 20 minutes. Store at RT.

• Trace elements solutions, 100x stock solution, per liter
  • 100 mg MnCl2.4H2O (Merck)
  • 170 mg ZnCl2 (Sigma)
  • 43 mg CuCl2.2H2O (Merck)
  • 60 mg CoCl2.6H2O (Merck)
  • 60 mg Na2MoO4.2H2O (Aldrich)

• • Contrary to what is described by Harwood and Cutting, MgSO4 and CaCl2 are added as separate solutions to prevent precipitation problems. FeCl3 is also added as a separate solution as this compound is unstable. Filter-sterilize the solution and store in the dark at RT.

• Calciumchloride solution (100mM, per 100 mL):
  • 1.47 g CaCl2.2H2O (Merck)
  • Autoclave and store at RT.

• Magnesiumsulfate solution (1M, per 100 mL):
  • 24.6 g MgSO4.7H2O (Merck)
  • Autoclave and store at RT.

• Iron chloride solution (50 mM in 100 mM citric acid, 100 mL):
  • 1.35 g FeCl3.6H2O (Sigma)
  • 2.10 g citric acid.H2O (Sigma)
  • Filter sterilize and store in the dark at 4oC.

• Glucose solution:
  • 500 g C6H12O6 (Merck)

(50% w/v, per liter) Dissolving the glucose takes time: use a warm waterbath to speed up the dissolving. Solution is autoclavable at 121oC for 20 minutes. Filter sterilization is also possible.

Or H) Glucose solution: 200 g C6H12O6 (Merck) (20% w/v, per liter) Dissolving the glucose takes time: use a warm waterbath to speed up the dissolving. Solution should be filter sterilized.

I) L-malate solution: 134 g C4H6O5 (Sigma) L-form!! (13.4% w/v, per liter) Use a 4M NaOH solution to adjust the pH of the solution to neutral (pH 6.0-8.0). Requires roughly 47ml 4M NaOH per 100ml of solution. Solution should be filter sterilized.

NOTE: For all solutions Nanopure H2O with a electric resistance of more than 17 MOhm/cm (= conductivity of less than 60 nS * cm) is used.

Preparation of the medium

• For 1 liter of M9 medium add in the following order:
  • 200 mL M9 5x stock (sol. C)
  • 1 mL 100 mM CaCl2 (sol. E)
  • 10 mL trace salts 100x stock (sol.D)
  • 1 mL 1 M MgSO4 (sol. F)
  • 1 mL 50 mM FeCl3/100 mM C6H8O7 (sol. G)
  • 6 mL of glucose 50% (w/v) stock solution (sol. I)

• • Or 15 mL of glucose 20 % or 15 mL of DL-malate 20% for 0.3 % carbone source
  • Or 10 mL glucose 50%, or 25 mL glucose 20%, or 25 mL DL-malate 20% for 0.5%

• • add nanopure H2O to a final volume of 1 liter