STEP 5. Sterilization in Pressure Cooker or Autoclave to eliminate all forms of microorganisms in substrate (molds, fungi, bacteria, spores). Time vs Pressure for sterilization process. Alternative thermal treatment in multicooker, instant pot and oven

Grain substrate sterilization before inoculation in mushroom growing
Step 5. Grain substrate sterilization before inoculation

STEP 5. Day 2. Grain substrate sterilization for mushroom spawn

At this stage, you've already have prepared substrate and jars for substrate with injection port and air filter. Now we need to execute substrate sterilization process.

What is sterilization?

Sterilization — heat treatment that deactivates all forms of microorganisms in substrate, such as bacteria and mold spores. It is carried out at temperatures above 212°F (+100°C) for a certain period of time.

You can buy ready for inoculation sterile grain for mushroom spawn, so that skip next steps and move on Inoculation section. Such option can save your time greatly and money for expensive pressure cooker, jars and lids accessories. It's a great option if you just want to try mushroom cultivation with ease.

Shroomok's choice on Amazon:

Sterilized Rye Berry Mushroom Substrate with Self Healing Injection Port - 1/6/12/25 Bags - $9.69

Sterilized Wheat Grain Bag with Injection Port - 3 Lb - 1 Bag - $16.88

Sterilized Rye Grain Berries for Mushroom Spawn | Quart Jars - 1 Jar - $17.99

Sterilized Rye Grain Mushroom Substrate in Injectable Jars - 2 Jars - $29.56

Sterilized Rye Berry Grain Bag with Injection Port - 3 lbs (2 Bags Each 1.5 lb) - $39.74

Sterilized Rye Berries Substrate in Mushroom Grow Bags - 1 Pound - 6 Bags - $43.95

Sterilized Rye Berry Mushroom Substrate in Injectable Quart Jars - 4 Jars - $53.95

BRF Jars Brown Rice Flour Mushroom Substrate - 6 Jars - $55.00

All you need to prepare for grain sterilization

1️⃣ Pressure Cooker, Autoclave, Pressure Multicooker or Instant Pot

Pressure Cooker for Grain Sterilization
Pressure Cooker for Grain Sterilization

Shroomok's Choice on Amazon:

Pressure Cooker 15-PSI, Polished Aluminum - 22 Qt $119.99

Pressure Cooker & Canner, 3 PSI Settings - 22 Qt $131.99

Pressure Canner and Cooker - 23 Qt $159.99

Professional All-American Non-Electric Sterilizer - 25 Qt - $449.95

2️⃣ Prepared grain substrate.

Prepared grain substrate
Prepared grain substrate

🔴 Check step Choose and prepare Substrate and Cooking the substrate

3️⃣ Prepared Spawn Jars or Spawn Bags with inoculation port and air filter

Spawn Jars with Injection Port and Air Filter
Spawn Jars with Injection Port and Air Filter
Spawn Bags
Spawn Bags

🔴 Check step: Jars and Bags for Mushroom Spawn

4️⃣ Vermiculite or Vermiculite/Perlite 50/50.

Vermiculite and Perlite
Vermiculite and Perlite

Shroomok's Choice on Amazon:

Vermiculite 8 qt. - $14.98

Organic Vermiculite 8 qt. - $15.39

Premium Perlite 8 qt. - $13.79

Organic Perlite 8 qt. - $16.89

5️⃣ Aluminium Foil.

6️⃣ Water.

Preparation for grain substrate sterilization

1️⃣ Add 1 table spoon of vermiculite on the bottom of each jar (just to cover the bottom). In case there will too much moisture in the substrate, this vermiculite will help to compensate. It will absorb the excess moisture when you shake the jar with substrate. Recommended for wheat berry and brown rice

Preparing for sterilization substrate. Add 0,5-1 cm of vermiculite on the bottom of each jar
Add 5-10 mm of vermiculite on the bottom of each jar

2️⃣ Fill the jars with the prepared substrate, but no more than 2/3 or 3/4 of the jar. In the future you'll need to shake the content of the jars without opening them. If you fill the jar to the top, you will not be able to shake the substrate. Additional space needed for gas exchange as well

Fill jars with prepared substrate for growing psilocybin mushrooms
Fill 2/3 of each jar with prepared substrate

3️⃣ Close the jars tightly with modified lid (lid with inoculation port and filter). During sterilization lids on the jars may loosen under the influence of temperature. This applies to both metal and polypropylene lids. Make sure they are tightly closed before sterilization

Jars with substrate for further sterilization
Jars with substrate for further sterilization. Fill jars on 2/3 or 3/4.
Sterilization preparation
Jars with substrate covered by lids with inoculation port and air filter

4️⃣ Wrap the lids of the jars with foil. So that water will not seep through the air filter on the lid during sterilization

Wrap lids (inoculation port and filter) with foil
Wrap lids with foil
Substrate ready for sterilization
Substrate and jars ready for sterilization

Grain sterilization in multicooker, pressure cooker, autoclave

1️⃣ On the bottom of the pressure cooker (or multicooker) put a metal stand or cloth napkin. The jars and the bottom should not touch each other and less banging in the multicooker. This more concerned to glass jars, so that they do not break during sterilization

Preparing pressure cooker (multicooker) for substrate sterilization
Preparing pressure cooker (multicooker) for substrate sterilization

❗️If the jar cracks or breaks during sterilization, you can't use it for further inoculation!

2️⃣ Fill the multicooker (or pressure cooker) with water. The level of water should reach half of the jars or a little bit more. Just keep in mind that the water will boil and if you pour more water, the lids will be filled with water. We can't let it happen!

Fill the multicooker (or pressure cooker) with water for sterilization substrate
Fill the multicooker (or pressure cooker) with water for substrate sterilization

3️⃣ Put the jars in pressure cooker (autoclave or multicooker). We put only 3 jars of 500 ml or 2 jars of 720 ml to it. Polypropylene jars can float because of their lightness. To prevent them from turning over, you can put a ceramic plate on top and close the lid of multicooker (pressure cooker).

Multicooker (pressure cooker) ready for sterilization substrate for cultivation psilocybin mushrooms Psilocybe Cubensis
Multicooker (pressure cooker) ready for sterilization substrate for growing mushrooms
Substrate sterilization in pressure cooker
Substrate sterilization in pressure cooker

4️⃣ Set the temperature +121°C or 250°F or 15 psi for Pressure Cooker and Autoclave.

Grain substrate sterilization for mushroom cultivation
Grain substrate sterilization in autoclave - 121°C or 250°F or 15 psi - for mushroom grain spawn

Sterilization time depends on the type of grain:

⏰ Wheat berry, oats, barley, rye berry, BRF substrate, brown rice - 1 hour 30 minutes

⏰ Millet - 1 hour 20 minutes

⏰ Corn, popcorn, sorghum - 2 hours

🔴 Pay attention! The sterilization temperature should be higher than +100°C or 212°F! It gives more chances to destroy all bacteria and their endospores that still alive in the grain. Sterilization is about bacteria prevention, because all mold spores die at 60°C or 140°F and higher during 30 minutes.

5️⃣ When sterilization is complete leave the substrate cool down slowly for at least 8 hours.

Don't open the PC/multicooker and don't take the jars out immediately after sterilization is complete. Why?

⛔️ You can suffer yourself with the steam.

⛔️ If you open the PC/Autoclave/Multicooker the glass jars can break because of the difference in pressure and temperature. Broken jars go straight to the trash. Polypropylene jars you can take out immediately, but it would be better to let them cool down a little without a sharp temperature changer

⛔️ Cold (not sterile air) come through air filter due to rapid temperature changes and pressure changes. It can provoke contamination in the future

6️⃣ When you take the sterile substrate out of the pressure cooker leave it to chill at room temperature. Jars/Bags can be warm a little bit. Do NOT remove the foil!

Sterile substrate ready for inoculation psilocybin mushroom spores
Sterile substrate ready for inoculation Psilocybe Cubensis spores

Mild condensation can appear in jars as they chill - don't worry, it's okay!

7️⃣ Repeat the same steps with other jars.

How to sterilize grain substrate for mushrooms without a pressure cooker or autoclave

How to make grain sterilization in pot on the stove

Load jars in pot.

Add water, water level is 1/2 of jars height.

Bring the water to boil.

Make heat treatment for 90-120 minutes.

Take out the jars. Let the grain cool down and wait for 24-48 hours.

Repeat heat treatment for the same jars with substrate for 90-120 minutes again.

Don't open the lid of pot. Let the jars cool down in pot.

Take out the jars. Be sure that jars cool down to room temperature.

Jars ready for inoculation!


One more option if you don't have pressure cooker is Tyndallization It is old school method and takes 3 days, but it works.

Tyndallization essentially consists of heating the substance to boiling point 100°C or 212°F and holding it there for 15-30 minutes, three days in succession. After each heating, the resting period will allow spores that have survived to germinate into bacterial cells; these cells will be killed by the next day's heating.

If you are going to try tyndallization keep in mind you should start counting time after jar/bag with substrate reach max temperature inside. If you put jar with spawn inside boiling water it will take some time before center of jars reach required temperature. Use at least 30 minutes treatment.

How to make grain sterilization in the oven

This method is worst than heat treatment in pot, because grain usually dry out in the oven. Sterilization in oven can be used for big grains like corn. Wheat, rye, oat become totally dry after oven tretment.For corn sterilization in oven add 1 table spoon of water in each jar and close the lid. Cover each jar with foil.

Preheat oven at 150°C or 300°F.

Then load the oven with the jars sideways to have thiner water level we added before (extra 1 table spoon of water).

Leave the jars in the oven at 150°C or 300°F for 30 minutes.

After 30 minutes set the temperature 120°C or 250°F and sterilize for another 2 hours.

Turn off the oven, take out the jars. Let them cool till room temperature for about 8 hours.

Jars ready for inoculation!

Useful notes

Does it possible to sterilize substrate without a pressure cooker (PC)? You can sterilize substrate in the oven. But PC/Autoclave is preferred because sterilization under pressure gives higher temperature in the substrate. Some mushroom growers sterilize jars with substrate just in a saucepan on the stove. This option is suitable for sterilizing secondary substrates (hay, coconut peat soil etc). Grain substrate requires higher sterilization temperatures to deal with bacteria. Therefore, in most cases, sterilization without a pressure cooker/multicooker leads to substrate contamination and all work done before will go to waste.

Make sure that the lids on the jars are tightly closed before and after sterilization. It can happen that metal lids come loose and need to be tightened. If the lid is open at all (this happens with polypropylene lids), then the substrate must either be discarded or sterilized again.

✅ Ready non-sterilized substrate in jars/bags can be stored in the refrigerator for 2-3 days.

✅ Ready sterilized substrate in jars/bags can be stored for 3-5 days before being inoculated.

When all jars cooled down to room temperature move on — STEP 6. Day 2. Inoculation 🔽

  1. Cultivation Guide
  2. Substrate sterilisation