Nov 06, 2025

Bacterial Liquid Carbon Free Media

DOI
https://dx.doi.org/10.17504/protocols.io.8epv5kmrdv1b/v1
  • 1University of Milan
Conor James Richard Scott
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DOI: https://dx.doi.org/10.17504/protocols.io.8epv5kmrdv1b/v1
External link: https://scottc-bio.github.io/CJRS-protocols/
Protocol Citation: Conor James Richard Scott, Davis Roma 2025. Bacterial Liquid Carbon Free Media. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5kmrdv1b/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 09, 2025
Last Modified: November 06, 2025
Protocol  Integer ID: 229429
Keywords: Microbiology, Minimal, Media, Bacterial, Carbon-Free, free minimal media for bacterial growth, bacterial growth, liquid culture, carbon source, basic carbon, added carbon source, different carbon source, base media, solid media, free minimal media, free media, agar for solid media, protocol for growth, media
Disclaimer
This is not an optimised media for growth. Just a carbon-free base.
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.
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.
Materials
Chemicals
Na2HPO4•5H2O
KH2PO4
NaCl
NH4Cl
FeSO4•7H2O
MnSO4•5H2O
CuSO4•5H2O
ZnSO4•7H2O
MgSO4•7H2O
CaCl2
Agar powder
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
Safety warnings
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 bottles when autoclaving!
Always wear gloves and goggles when handling the 0.1 M HCl.
10X M9 Salts
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
Add 400 mL dH2O and stir with a magnetic stirrer until dissolved
Once fully dissolved, make up to 500 mL with dH2O
In a laminar flowhood, filter sterilise the solution using a sterile syringe and 0.22 μm pore filters into an autoclaved 500 mL bottle.
1000X Trace metals
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
Add 40 mL 0.1 M HCl and stir with a magnetic stirrer until dissolved
Once fully dissolved, make up to 50 mL with 0.1 M HCl
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 MgSO4
Add the following to a 100 mL beaker:
  • 12.3 g MgSO4•7H2O
Add 40 mL dH2O and stir with a magnetic stirrer until dissolved
Once fully dissolved, make up to 50 mL with dH2O
Move into a sterilised laminar flow hood and filter sterilise through 0.22 μm pore using a syringe into a sterile falcon tube
1M CaCl2
Add the following to a 100 mL beaker:
  • 7.35 g CaCl2
Add 40 mL dH2O and stir with a magnetic stirrer until dissolved
Once fully dissolved, make up to 50 mL with dH2O
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)
Combine the following in a 500 mL bottle:
  • 40 mL 10X M9 Salts
  • 0.4 mL 1000X Trace Metals
Make up to 400 mL with dH2O
Autoclave
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
Swirl to mix and the media is now ready for use.