Date updated: 2025-11-05

Author: Conor JR Scott, Davis Roma

Affiliation: Università degli Studi di Milano

Version: 1.2

Related DOI: View the published protocol at protocols.io

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.

Materials & Equipment

Chemicals

  • FeSO4•7H2O
  • EDTA•2Na (EDTA di-sodium salt)
  • 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

Method

1000X Hutner’s Trace Elements

Solution 1

  1. Combine the following in a 100 mL glass beaker:
    • 1 g FeSO4•H2O
    • 12.7 g EDTA•2Na

  2. Add 80 mL dH2O

  3. While continuously stirring with a magnetic stirrer use KOH pellets to increase the pH to a maximum of 6.5 or until any last solute dissovles fully to leave a golden-yellow solution

Solution 2

  1. Combine the following in a 100 mL glass 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)

  2. Add 80 mL dH2O and stir with a magnetic stirrer until fully dissolved

  3. Combine solutions 1 and 2 in a 250 mL bottle

  4. 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

  5. Bring the final volume up to 200 mL with dH2O

  6. 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

  1. Combine the following in a 500 mL beaker:
    • 60 g NaNO3
    • 5.2 g KCl
    • 8.1 g KH2PO4
    • 10.5 g K2HPO4

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

  3. Once dissolved, make up to 500 mL and autoclave

  4. Store at 4 °C

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

Preparing Liquid Carbon Free Media (LCFM)

  1. Combine the following in a 500 mL duran bottle:
    • 20 mL 20X fungal minimal salts
    • 0.8 mL 1 M MgSO4
    • 0.4 mL Hutner’s trace elements

  2. Make up to 400 mL with dH2O

  3. Autoclave and allow to cool

  4. The media is now ready for liquid cultures

Note: After autoclaving a white precipitant may be visible, but this will dissapear upon cooling.

Additional notes

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

Keywords

Microbiology, Minimal, Media, Fungal, Carbon-Free

Disclaimer

This is not an optimised media for growth. Just a 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 eye protection when using 1 M KOH

Version History

Version Date Changes
1.0 2025-10-10 Initial creation of protocol
1.1 2025-10-23 Re-ordered to focus on materrials and methods; Changed recipe to EDTA di-sodium salt; Improved notes on solution 1 pH adjustment
1.2 2025-11-05 Reduction of volumes to be more reasonable