Fungus Inoculation of a liquid culture

Tutorial de avatarLow Tech Developer | Categories : Food, Water

Discover how easy it is to grow mushrooms! Easy and quick to set up, liquid inoculation consists of depositing a liquid culture on a substrate. The mycelium will propagate during the incubation phase. It is then possible to move on to the fruiting phase to pick the mushrooms.

License : Attribution (CC BY)


In this tutorial, we are going to learn how to use a mycelium syringe in liquid culture. One of the prerequisites is therefore to have a syringe of liquid culture of the mushroom variety of your choice. It is possible to provide it on the link following. Tutorial produced in partnership with the company Breizh Bell, a company in myciculture and low-tech production of organic mushrooms, in France.

Introduction to the mushroom cycle

What is commonly called "mushroom" is in fact only the temporary and visible "fruiting body": the sporophore (formerly called "carpophore").

It is the result of an organism with a more durable and more discreet character, formed of filaments generally invisible to the naked eye when they are isolated: the A9lium mycelium.

The sporophore is often in the form of a foot wearing a hat .

To cultivate a mushroom, we try to reproduce the natural external conditions. Mushroom cultivation is divided into three main stages.

  1. Inoculation: addition of mycelium (in the form of spores or in liquid or agar culture) in the substrate
  2. Incubation: the mycelium propagates in the substrate
  3. Fructification: the mycelium wants to reproduce by forming a sporophore

Liquid inoculation?

In myciculture, inoculation by liquid culture consists of injecting mycelium contained in sterilized sugar water onto a substrate.

Why is it awesome?

There are several ways to start a mushroom crop. Liquid inoculation is one of the easiest because you just need to spray a substrate with a liquid culture. In addition, the risk of contamination is less than with other methods.

Receiving the liquid inoculation syringe

Upon receipt of the syringe containing the mycelium, place it immediately in the refrigerator, it must be used within two weeks!

You have two choices :

  1. Liquid culture copy: The goal is to copy the liquid culture to achieve even more culture! The liquid culture copy is optional!
  2. Grain inoculation: This consists of depositing a liquid culture on a substrate. Seed inoculation can be done in a jar or in a bag
When copying liquid culture, you can only make this copy once. The mycelium weakens with the copies and your crops will not produce correctly.

Glossary :

Mycelium: filamentous body of the fungus

Inoculation: injection of the mycelium into the substrate

Liquid culture: mixture of sterilized sugar water with mycelium

Substrate: Food of the fungus (grains, straw, wood...), the mycelium attaches to the substrate

Video overview


Liquid culture

(Liquid Mycelium syringe: Namekos, blue oyster mushroom, gray oyster mushroom, yellow oyster mushroom, pink oyster mushroom, shiitake mushrooms, red wine stropharia, etc.)

Kit Laborantin

– Pairs of talc-free gloves and masks.

– Charlotte or clean cap (the swimming pool ones are tops!).

– Tealight candles (Minimum 10 to 20)

- Sterile compresses

Lab equipment

- Paper towels

– Alcohol 70°

- Sacs de culture

Equipment for liquid culture:

- A jar and its lid

- A drill and drill bits

- Un bouchon auto refermable & un bouchon filtrant

- High temperature transparent silicone seal tube

- Aluminum foil

- water and beet sugar


– Thermometer

- Measuring cup

- Pair of scissors

– Clothespins (wooden recommended)

– Large saucepan/pressure cooker or oven.

– Vaporizer

Step 1 - Preparation

It is very important to have the most sterile conditions possible, throughout the manipulations. Indeed, there is a biological struggle between living species. If the environment is contaminated, the fungus will find itself in competition with other organisms, so it will be too fragile and will lose. We will seek to create ideal growing conditions to promote the fungus.

We advise you to place yourself in your kitchen, under your hood. It will prevent dust from settling on your crops.

- Clean your work surface and each potential contact area with 70° alcohol before each handling.

- Wash your hands and arms thoroughly with soap before each handling. You can also put on gloves and clean them with 70° alcohol.

- Tie your hair and/or put on your hat.

We strongly advise you to handle your liquid mycelium out of sunlight, under a soft and weak light (night light for example).

Step 2 - Optional, copy of a liquid culture: manufacture of the jar for inoculation

In this step, we will make a jar for your liquid culture.

The lid of the jar will have two holes. They will be filled with two plugs.

The first stopper is self-closing and will allow the insertion of the syringe needle. The second is a filter cap for the respiration of the mycelium in the jar.

- Take a jar and make the two holes using a drill (be careful, the diameter is not the same!).

- Place a light dose of silicone gasket all around the hole of the filter cap

- Place the plugs in the holes with the appropriate diameters.

- Let the seal dry for a day

Your jar is ready for liquid inoculation!

Step 3 - Optional, liquid culture copy

- Follow step 1 of this tutorial.

- Boil water in your saucepan.

- Take your previously prepared jar (see step 2), fill it with boiled water and unrefined beet sugar (volume 4% of the volume of water). Close your jar, cover it with aluminum foil, emphasizing the opening area of the lid.

- Fill your pot with water again and boil it.

- Place your wrapped jar in your saucepan and leave it covered for 2 hours (sterilization).

- In your kitchen, turn on your hood and clean your worktop with alcohol.

- Place tealights all around your work area.

- Light the candles and take your syringe of liquid mycelium out of the refrigerator, clean (alcohol 70°) and place it in the center of the work area.

- Take your syringe and needle out of their bag. Insert the needle.

- Take your jar of liquid culture, clean the outside of this jar with 70° alcohol and place it in your work area.

- Take two sterile compresses and soak them in alcohol.

- Take two sterile compresses and soak them in alcohol.

- Then place it between the two compresses soaked in alcohol.

- Completely empty the mycelium from your syringe into your jar.

- Wrap your jar with aluminum foil, fabric or paper

- Place it in an environment at 20°C and wait about two weeks. You can shake your jar to make your mycelium spread even more and take the opportunity to see its evolution
Watch out for the light!

The mycelium will spread.

Your culture is good when your jar is filled with a cloud of mycelium! The slightest green, blue or black stain in your jar is a sign of contamination and your culture will then be unusable.

When the mycelium has spread well, we invite you to carry out an inoculation on the grains. The steps are described below

Step 4 - Grain inoculation: the rinse, day 1

If you want to inoculate grains, they will have to be cleaned on the first day.

- Take out your grains and rinse them thoroughly with cold water, until clear water is obtained.

- Place these grains in a container.

- Take calcium carbonate (CaCO3) and add 2 to 5% of the weight of your grains.

- Fill this bag with hot tap water, slightly above the level of the beans.

- Close your container and let stand overnight.

Step 5 - Grain inoculation: sterilization, day 2

- Boil water in your saucepan.

- Take out your container of beans and fill it with boiled water (below the level of the beans).

- Fill your vaporizer with the rest of this cooled water.

- Fill your pot with water again and bring to the boil.

- Close the container of your grains and immerse it in your saucepan/pressure cooker.

- Let it boil for 3 hours in your pan, covered.

- Take your container out of the pan and let it cool overnight.

Step 6 - Grain inoculation: day 3

The liquid inoculation is carried out on the clean substrate. Either the substrate is in a jar (as in the pictures) or the substrate is contained in a grow bag. In both cases, the mycelium must be able to breathe.

If you choose a jar, pierce its lid and clean it with 70° alcohol. Then place a sterile compress in the hole.

- In your kitchen, clean your worktop with alcohol.

- Turn on your cooker hood and place tealights all around your work area.

- Light the candles and take your syringe of liquid mycelium out of the refrigerator, clean (alcohol 70°) and place it in the center of the work area.

- Take your syringe and needle out of their bag. Insert the needle.

- Take your bag/jar of sterilized grains, clean the outside of this bag/jar with 70° alcohol and place it in your work area.

(Be careful that the filter/compress does not touch alcohol)

- Take two sterile compresses and soak them in alcohol.

- Place the needle of your syringe on the flame of one of the candles and wait for the tip to turn red.

- Then place it between the two compresses soaked in alcohol.

- Open your bag/jar in the work area and completely empty the mycelium from your syringe onto your seeds.

- Close the bag/jar immediately by folding the top and attaching your clothespins.

- Expel the air and place it in a non-transparent box at the incubation temperature adapted to the variety (information on the Breizh Bell website).

- Wait for the incubation time adapted to the variety (14 days oyster mushrooms, other information on the Breizh Bell website). Write it down in your diary!

(you will be able to see if your mycelium is colonizing your grains correctly after a few days, but be careful with the light!).

The mycelium will spread.

Your crop is ready when your beans turn completely white. The slightest green, blue or black stain in your jar is a sign of contamination and your culture will then be unusable.

Step 7 - Seed transfer: cloning or fruiting

As soon as your grains are colonized, you have the following two options:

  1. Transfer your colonized grains to other grains for copy
  2. Transfer your colonized seeds to a substrate for fruiting

It is advisable to copy the mycelium no more than three times. As with the liquid culture copy, the mycelium weakens with the number of copies.

Step 8 - Preparation of your substrate (as soon as your grains are well colonized)

Attention, the following steps are only valid for oyster mushrooms!

This step will allow the already stronger mycelium to settle on a harder substrate. Oyster mushrooms are fond of sawdust from hardwoods or straw. You can also combine the two.

Natural (non-odorized) rabbit litter is an ideal substrate and easy to find in stores!

- Heat water in your saucepan over low heat, you can add coffee grounds (maximum 1/3 of the volume of the substrate and maximum three days) to this water!

- Take a container and fill it with hardwood sawdust and/or straw.

- Take your thermometer and wait for the hot water to reach 75°C (pasteurization).

- Fill your bag of substrate with this water and reheat the water over low heat.

- Preheat your oven to 75° if you find that your container of substrate is too large for your pan!

- Take your thermometer, and measure the temperature of the water, it should never exceed 93°C!

- To know if your substrate is well moistened, take a good handful of it in your hand and squeeze it. It should come off on its own and not stay in a ball. For a better understanding, we invite you to watch the tutorial video “Wet your substrate well!

- Fermez votre contenant (à l’aide de pinces à linge en bois en cas de sac).

- Plongez votre contenant dans l’eau, laissez-le pendant 2 heures à couvert et prenez la température de l'eau chaque demi-heure (<93°C) // Placez votre contenant dans votre four à 75°C pendant deux heures !

- Laissez-les refroidir une nuit et conservez-les fermés.

Step 9 - Transfert des grains

Dans votre cuisine, nettoyez votre plan de travail à l’alcool.

- Allumez votre hotte et placez des bougies chauffe-plat tout autour de votre zone de travail.

- Prenez votre contenant de grains inoculés, le mycélium doit avoir coloniser l’ensemble des grains !

- Nettoyez l’extérieur du contenant de grains inoculés et le contenant de substrat refroidi avec de l’alcool.

- Ouvrez vos contenants au centre de la zone de travail.

- Cassez vos grains colonisés pour en faire des grumeaux, attention à ce que chaque instrument en contact soit nettoyé !

- Remplissez votre contenant de substrat avec vos grains inoculés. Fermez-le immédiatement (avec les pinces à linge, en chassant l’air en cas de sac).

- Replacez les contenants dans une pièce à une température comprise entre 20 et 23°C pendant trois semaines. Prêt d’un radiateur dans votre salle de bain est une solution astucieuse !

En cas de sac, placez le dans une boite isolée de la lumière.