How to make Petri dishes for mycological culture: Beginner's guide

Mushroom cultivation and mycology, the study of fungi, are fascinating fields attracting more and more enthusiasts, both amateurs and professionals.

For those wishing to start mushroom cultivation in the laboratory or at home, using Petri dishes is an essential starting point.

This practical guide explains why to use Petri dishes, how to choose the right culture medium, and presents a simple method to prepare and pour agars for your mycology experiments.

Why use Petri dishes in mycology?

Petri dishes are flat, transparent containers, usually made of plastic or glass, mainly used to cultivate microorganisms on nutrient agar.

In mycology, they create a controlled environment to observe and study fungal growth, whether for research, cloning, strain selection, or simply learning. Petri dishes offer several advantages:

1. Control of culture conditions: They allow manipulation of parameters such as medium type, temperature, humidity, and light exposure.
2. Easy observation: The transparent lid facilitates direct observation of mycelium development without opening the dish, thus minimizing contamination risks.
3. Ease of isolation and selection: Petri dishes allow isolating specific strains or selecting healthy mycelia for future cultures.

Different culture media for Petri dishes and their uses

The choice of culture medium is crucial in mycology, as it directly affects the growth and health of fungi. Culture media come in powder form to be mixed with water and are enriched with various nutrients to promote fungal growth. Here are the main types of culture media and their uses:

1. MEA+ medium (Malt Extract Agar + Peptone)

   Rich nutrient medium, suitable for cultivating most mycelia. It also promotes the development of bacteria, making it a good choice for testing air or sample purity (contamination tests).

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   Medium "Sabouraud + Antibiotic"

   Enriched with an antibiotic (usually chloramphenicol), this medium limits bacterial growth and becomes very selective. It is ideal for sensitive manipulations such as cloning wild fungi or spore germination, especially under non-sterile conditions. However, mycelial growth may be somewhat slower than with a medium like MEA+.

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   Medium "Water Agar" (WA)

   Pure agar without added nutrients. Used for:

   • the strain purification from contaminated tissues,

   • the natural selection (only fungi survive),

   • specific observations (morphology, slow growth tests). It is a neutral support, useful as a transitional step.

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   Medium "PDA" (Potato Dextrose Agar)

   Prepared from potato extract and glucose, it is a classic and universal medium in mycology. It stimulates mycelium growth and allows good observation.
   Used for:

   • the general mycelium culture,

   • the strain preservation,

   • the biomass production.
     However, it is sensitive to contamination due to its richness in sugars.

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   Medium "MYA" (Malt Yeast Agar)

   Contains malt extract and yeast. It is particularly suited for:

   • the spore germination,

   • the demanding species (such as Hericium erinaceus or certain forest species),

   • the conditions where vigorous growth is sought.
     It can be enriched with peptone to become a very complete medium (MYPA).

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   "GYEA" (Glucose Yeast Extract Agar) medium

   A simple but nutritious medium, composed of glucose, yeast extract, and agar.
   Advantages:

   • fast growth,

   • easy to adapt formulation,
     but less used in routine culture than MEA or PDA.

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   "Corn Meal Agar" (CMA) medium

   Less rich, often used to observe the morphology of reproductive structures (such as spores or specialized hyphae).
   Rarely used in production, but useful in laboratory or taxonomic identification.

You can also get our sterile Petri dishes, perfect for these applications, here.

Recipe for preparing MEA or Sabouraud agar

Preparing an agar is an essential step for culturing mushrooms in Petri dishes. Here is how to easily make an MEA or Sabouraud culture medium:

Necessary ingredients:

• 47 g of MEA or Sabouraud medium powder.
• 1 liter of hot water (preferably distilled or demineralized).

Preparation steps:

1. Mixing : Dissolve 47 g of medium powder in 1 liter of hot water. Mix well to ensure the powder is completely dissolved.

2. Sterilization : Sterilize the mixture at 121°C (15 PSI) for 20 to 30 minutes. This can be done using an autoclave or a pressure cooker. This step is crucial to eliminate any unwanted microorganisms.

3. Cooling : Let the mixture cool down to about 45-50°C. This cooling is important to prevent condensation from forming on the lids of the Petri dishes.

4. Pouring: In a sterile environment (under a laminar flow hood or near a Bunsen burner), pour the agar into the Petri dishes. Ensure the agar thickness is about 0.5 to 1 cm.

5. Closing: Quickly close Petri dishes to avoid contamination from ambient air.

Sterility Techniques in Mycology

Sterility is one of the most crucial factors in mushroom cultivation, as contaminants like bacteria and molds can quickly invade your cultures and ruin your work. In mycology, establishing a sterile environment throughout the cultivation process is therefore essential to obtain reliable results. Here are some essential sterility techniques to minimize contamination risks.

1. Using a laminar flow hood

A laminar flow hood is equipment designed to provide a sterile environment by filtering air and pushing it in a uniform direction. This limits the circulation of contaminated air over work surfaces. If you do not have a hood, you can work near a Bunsen burner flame, which creates a sterile air barrier.

2. Wearing gloves and a mask

Before any handling, it is recommended to wear sterile gloves and a face mask. Disinfect your gloves with 70% isopropyl alcohol before starting each handling. This reduces the possibility of transferring contaminants from hands to cultures.

3. Sterilization of tools

All tools used, such as scalpels, tweezers, or needles, must be sterilized. Using a Bunsen burner to sterilize these instruments by heat is a common method. It is also possible to sterilize them with 70% isopropyl alcohol followed by a flame to burn off alcohol residues.

4. Sterilization of culture media

Culture media (agar, liquids) must be sterilized before use. The most common method is to autoclave or pressure cook them at 121°C for 20 to 30 minutes to eliminate all unwanted microorganisms.

5. Disinfection of the work surface

The work surface must be carefully cleaned and disinfected before and after each work session. A 70% alcohol spray or a diluted bleach solution can be used to disinfect surfaces, dishes, and other materials to be handled.

6. Minimal opening of Petri dishes

When working with Petri dishes, it is important to open them as little as possible and keep their opening facing the sterile airflow or the Bunsen burner flame to avoid contamination from ambient air.

Typical Example: Collecting a Carpophore from Nature for Cultivation

Here is an example detailing the steps to follow to collect a carpophore (visible part of the mushroom) from nature, cultivate it on Sabouraud + chloramphenicol agar for cloning, then multiply it on MEA agar, and finally perform a liquid culture.

Step 1: Preparation of Culture Media

• Prepare a Sabouraud + chloramphenicol agar according to the instructions described in the previous article.
• Also prepare an MEA agar that will be used later for mycelium multiplication.

Step 2: Carpophore Sampling

• Find a healthy carpophore in nature.
• In a sterile environment, clean the surface of the carpophore with a cotton swab soaked in isopropyl alcohol to eliminate most external contaminants. Tear the mushroom while avoiding touching the internal parts of the mushroom.
• Using a sterilized scalpel, cut a small piece of the internal tissue of the mushroom. Work quickly to minimize exposure to air.

Step 3: Inoculation on Sabouraud + Chloramphenicol Agar

• In a Petri dish containing Sabouraud agar enriched with chloramphenicol (to limit bacterial growth), place the tissue piece taken.
• Quickly close the Petri dish and place it in an incubator at an appropriate temperature (usually around 25-27°C for most mushroom species).
• Regularly monitor the dish to observe the growth of the mycelium, which should appear as white filaments.

Step 4: Multiplication on MEA Agar 

• Once the mycelium is well developed on the Sabouraud agar, transfer a piece of this healthy mycelium using a sterile scalpel to a new Petri dish containing MEA agar.
• This transfer accelerates the growth of the mycelium in a more nutritious medium.

Step 5: Preparation of a Liquid Culture

• When the mycelium has spread well on the MEA agar, it is time to move to a liquid culture for a larger biomass production.
• Prepare a sterile liquid culture medium, such as malt broth, by sterilizing it in an autoclave.
• In a bioreactor or sterile bottle, inoculate this medium with a piece of mycelium taken from the MEA agar.
• Place the bottle in a dry, dark environment at an appropriate temperature and shake it regularly to ensure good aeration of the medium.