Nov 06, 2025

Fungal Liquid Carbon Free Media

DOI
https://dx.doi.org/10.17504/protocols.io.14egnrjwml5d/v1
  • 1University of Milan
Conor James Richard Scott
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DOI: https://dx.doi.org/10.17504/protocols.io.14egnrjwml5d/v1
External link: https://scottc-bio.github.io/CJRS-protocols/
Protocol Citation: Conor James Richard Scott, Davis Roma 2025. Fungal Liquid Carbon Free Media. protocols.io https://dx.doi.org/10.17504/protocols.io.14egnrjwml5d/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 10, 2025
Last Modified: November 06, 2025
Protocol  Integer ID: 229499
Keywords: Microbiology, Minimal, Media, Fungal, Carbon-free, fungal liquid carbon free media, free minimal media for fungal growth, fungal growth, liquid culture, carbon source, basic carbon, added carbon source, base media, different carbon source, free minimal media, agar for solid media, solid media, free media, media, protocol for growth
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 fungal growth. This is not an optimised protocol for growth, it is the base media to which different carbon sources can be added to detect fungal 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 can use what other glass vessels for mixing or autoclaving that you want.
Materials
Chemicals
FeSO4•H2O
EDTA
ZnSO4•7H2O
H3BO3
MnCl2•4H2O
CoCl2•6H2O
CuSO4•5H2O
(NH4)6Mo7O24•4H2O (Ammonium molybdate)
KOH pellets
1 M KOH
NaNO3
KCl
KH2PO4
K2HPO4
MgSO4•7H2O
dH2O

Glassware
100 mL beaker
250 mL bottle
500 mL beaker
500 mL bottle

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)
pH probe
Laminar flow hood (if unavailable, then can use a bunsen burner and good sterile technique around the sphere of influence)
P1000 + 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 duran bottles when autoclaving!
Always wear gloves and eye protection when using 1 M KOH
1000X Hutner's Trace Elements
Prepare Solution 1
Combine the following in a 100 mL glass beaker:
  • 1 g FeSO4•H2O
  • 12.7 g EDTA•2Na
Add 80 mL dH2O and stir with a magnetic stirrer until fully dissolved
Prepare Solution 2
Combine the following in a 100 mL glass duran/beaker:
  • 4.4 g ZnSO4•7H2O
  • 2.2 g H3BO3
  • 1 g MnCl2•4H2O
  • 0.32 g CoCl2•6H2O
  • 0.32 g CuSO4•5H2O
  • 0.22 g (NH4)6Mo7O24•4H2O (Ammonium molybdate)
Add 80 mL dH2O and stir with a magnetic stirrer until fully dissolved
Combine solutions 1 and 2 in a 250 mL bottle
Adjust the pH with KOH to pH 6.5 by initially using KOH pellets, and then 1 M KOH to gradually increase the pH to the final 6.5
Bring the final volume up to 200 mL with dH2O
Cover the bottle with foil and store at 4 °C, during storage the solution will turn from bright green to purple which is normal
20X Fungal Minimal Salts
Combine the following in a 500 mL beaker:
  • 60 g NaNO3
  • 5.2 g KCl
  • 8.1 g KH2PO4
  • 10.5 g K2HPO4
Add 400 mL dH2O and stir with a magnetic stirrer until fully dissolved
Once dissolved, make up to 500 mL and autoclave
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
Preparing Liquid Carbon Free Media (LCFM)
Combine the following in a 500 mL bottle:
  • 20 mL 20X fungal minimal salts
  • 0.8 mL 1M MgSO4
  • 0.4 mL Hutner's trace elements
Make up to 400 mL with dH2O
Autoclave and allow to cool
The media is now ready for liquid cultures