Date updated: 2025-11-05

Author: Conor JR Scott, Davis Roma

Affiliation: Università degli Studi di Milano

Version: 1.3

Related DOI: View the published protocol at protocols.io

Abstract

A simple protocol for the creation of a basic carbon free minimal media for bacterial growth. This is not an optimised protocol for growth, it is the base media to which different carbon sources can be added to detect bacterial growth and therefore metabolism of added carbon sources. The media can be used for liquid cultures, or combined with agar for solid media.

Materials & Equipment

Chemicals

  • Na2HPO4•5H2O
  • KH2PO4
  • NaCl
  • NH4Cl
  • FeSO4•7H2O
  • MnSO4•5H2O
  • CuSO4•5H2O
  • ZnSO4•7H2O
  • MgSO4•7H2O
  • CaCl2
  • 0.1 M HCl
  • dH2O

Glassware

  • 500 mL beaker
  • 500 mL bottle
  • 500 mL bottle (autoclaved)
  • 100 mL beaker

Plastic

  • Sterile 50 mL falcon tubes
  • Sterile syringes (largest size possible)
  • 0.22 μm pores for filter sterilisation

Equipment

  • Magnetic stirring equipment (not completely necessary, can stir/shake manually)
  • Laminar flow hood (if unavailable, then can use a bunsen burner and good sterile technique)
  • P1000 pipette + tips (sterile)
  • P100 + tips (sterile)
  • Autoclave
  • Aluminium foil

Method

10X M9 Salts

  1. Add the following salts to a 500 mL beaker:
    • 27.5 g Na2HPO4•5H2O
    • 7.5 g KH2PO4
    • 1.25 g NaCl
    • 2.5 g NH4Cl

  2. Add 400 mL dH2O and stir with a magnetic stirrer until dissolved

  3. Once fully dissolved, make up to 500 mL with dH2O

  4. In a laminar flowhood, filter sterilise the solution using a sterile syringe and 0.22 μm pore filters into an autoclaved 500 mL bottle.

Note: Filter sterilisation of the concentrated salts avoids the formation of white crystals that can occur during autoclaving. This maintains the clarity of the solution which is useful for minimal media where a single carbon source is added as it allows the clear detection of bacterial growth by eye.

1000X Trace metals

  1. Add the following to a 100 mL beaker:
    • 0.14 g FeSO4•7H2O
    • 0.12 g MnSO4•5H2O
    • 0.12 g CuSO4•5H2O
    • 0.14 g ZnSO4•7H2O

  2. Add 40 mL 0.1 M HCl and stir with a magnetic stirrer until dissolved

  3. Once fully dissolved, make up to 50 mL with 0.1 M HCl

  4. Move into a sterilised laminar flow hood and filter sterilise through 0.22 μm pore using a syringe into a sterile falcon tube

Note: The solution will be light blue in colour, for long term storage keep the tube wrapped in aluminium foil at 4 °C. If brown precipitate forms over time, then re-make the solution.

Note: The trace metals are also optional for the minimal media, the provide common cofactors for some enzymes which may be of interest.

1 M MgSO4

  1. Add the following to a 100 mL beaker:
    • 12.3 g MgSO4•7H2O

  2. Add 40 mL dH2O and stir with a magnetic stirrer until dissolved

  3. Once fully dissolved, make up to 50 mL with dH2O

  4. Move into a sterilised laminar flow hood and filter sterilise through 0.22 μm pore using a syringe into a sterile falcon tube

1 M CaCl2

  1. Add the following to a 100 mL beaker:
    • 7.35 g CaCl2

  2. Add 40 mL dH2O and stir with a magnetic stirrer until dissolved

  3. Once fully dissolved, make up to 50 mL with dH2O

  4. Move into a sterilised laminar flow hood and filter sterilise through 0.22 μm pore using a syringe into a sterile falcon tube

Preparing Liquid Carbon Free Media (LCFM)

  1. Combine the following in a 500 mL duran bottle:
    • 40 mL 10X M9 Salts
    • 0.4 mL 1000X Trace Metals

  2. Make up to 400 mL with dH2O

  3. Autoclave

  4. Once cool, in a sterilised laminar flow hood add the following with sterile pipette tips:
    • 0.8 mL 1 M MgSO4
    • 80 μL 1 M CaCl2
  5. Swirl to mix and the media is now ready for use.

Note: Adding MgSO4 prior to autoclaving will result in a cloudy white precipitant that does not re-dissolve. Adding CaCl2 after autoclaving might initially result in a small white preicpitant, but the volume is so small that swirling quickly re-dissolves it to achieve a completely clear bacterial minimal media.

Additional notes

To make minimal media agar, just add 6 g of bacteriological agar to the LCFM prior to autoclaving.

Keywords

Microbiology, Minimal, Media, Bacterial, Carbon-Free

Disclaimer

This is not an optimised media for growth. Just a clear carbon-free base.

Guidelines

Can adapt any of the volumes for your requirements. Also you can use whatever other glass vessels for mixing or autoclaving that you prefer or are available.

Warning

Always remember when autoclaving to use a larger glass vessel than the volume you want to autoclave to avoid it boiling over. Always remember to leave the bottle caps loose on duran bottles when autoclaving! Always wear gloves and goggles when handling the 0.1 M HCl

Version History

Version Date Changes
1.0 2025-10-10 Initial creation of protocol
1.1 2025-10-10 Added a note on the inclusion of trace metals
1.2 2025-10-23 Re-ordered to focus on materials and methods
1.3 2025-11-05 Reduced volumes to be more reasonable and solved precipitation issues