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July 8, 2022

Ultimate guide to sterilising mushroom substrate

An in-depth - but simple guide - exploring techniques used in preparing mushroom substrate. Focused on methods used for gourmet mushrooms.

This article explores 5 ways of sterilising mushroom substrate. It's ideal for the cultivation of any wood decomposing mushrooms such as:

  • Oyster Mushrooms
  • Shiitake
  • Ennoke
  • Lions Mane
  • Turkey Tail

It does not cover mushrooms growing in soil (field mushrooms), which mainly uses composting, however many of the concepts here a relevent.

1. Steam sterilisation

Ideal for

  • Lab equipment (scalpels, scissors etc)
  • Making plates, Petri dishes with Agar Agar
  • Grain spawn stages
  • Smaller 2-3kg bags, often called fruiting blocks.
  • Slower growing varieties such as Shiitake
  • Prefered by medium to advanced mushroom growers

The process

This process involves a careful relationship between Steam, Pressure, Time, and Temperature.

A) Pressure

At sea level, water boils at 100°C. No matter how much longer it is boiled, or by turning up the heat, it will not go above 100°C. This temperature is not sufficient in killing all pathogens. Bacteria like B. stearothermophilus, is only invalidated at temperatures above 121° C - as are many spores or endospores of fungi & bacteria. Raising the pressure is required to raise the temperature.

B) Temperature

The temperature required by most Mushroom growers is 121°C (250°F) requiring 15psi. Some equipment can go higher, so requiring less time.

0psi=100°C | 5psi=110°C | 10psi=116°C | 15psi=121°C | 20psi=26°C | 30psi=135°C

Pressure and temprature relationship

Note: at altitude, the water will boil at a lower temperature. So Higher pressure or a longer cycle will be required.

C) Time

The time required will vary depending on how much mass is loaded into the steriliser. The high temperature must reach the innermost part of the bag, so time is adjusted to compensate for the thermal mass to be heated. 10kg of substrate will require more time than 2kg. A typical load of a substrate, say 18-20kg, would require at least 3 hours. For a light load, say petri dishes, 15-30min should be sufficient. This does not include time to bring it up to pressure/temperature.

Using pressure sterilisation for mushroom substrate
Temperature and Time relationship

D) Steam

Moist heat destroys microorganisms by the irreversible coagulation and denaturation of enzymes and structural proteins. Dry heat is less effective. Substrate that is in bags should be pre-soaked, so moist. Heat outside the bag will raise the heat internally, causing the moisture in the bag to generate the steam within. If the substrate inside the bag is dry, this action will not take place, making it less effective.

Maintaining aseptic conditions

Once sterilised, aseptic conditions will be required for all stages going forward. It’s worth remembering, that the substrate is now the perfect habitat for the fastest organism to claim it. There are many fungi (moulds and yeast) plus bacteria who will colonise it more quickly than the culture we wish to grow.

Time required

Dependent on thermal mass.

  • Heating cycle 10-60+ minutes
  • Sterilisation cycle: 15min-3+ hours
  • Cooling cycle: 1-24 hours
  • Total time: 2 to 24 hours
Using pressure cooker for mushroom growing
Stages of Pressure Sterilisation

Equipment required

A) Retorts

These are often used in industry to process food items (like canned food). They are large, sometimes loaded with forklifts, and expensive. However, some commercial mushroom growers use them. A benefit is that they can be loaded from one end, outside, with the exit end in the clean-room itself. The large size translates to an equally large energy cost to run and labour required.

B) Autoclaves

These are often found in commercial labs and hospitals. They are simple and fast to operate. They are also very expensive, due to their automation and certification required. For mushroom growers most lab style autoclaves are too small.

However, All American produces one that qualifies as an autoclave rather than a pressure canner. It is the preference for many mushroom growers. A main differences between an autoclave and pressure cookers is that they maintain a vacuum during the cooling cycle and heating controlled by a thermostat.

C) pressure canners

Pressure canner is similar to a pressure cooker but tend to be larger and have less 'gimmicky' features. Making them simple and more practical to operate. An example would be the 'canners; from All American and Presto. Another benefit is that many pressure canners have the option to run on a hotplate so can be controlled with a thermostat (requiring a hack). For off-grid situations they can run on LPG/propane, or even a fire. A drawback is that the vacuum created at cooling draws unsterilised air back into the container.

D) pressure cookers

These are generally smaller and often have additional features (settings for different meals or steaming rice) that make them unappealing for use by mushroom cultivators. However, many people start on one of these. Although less practical that the ones above, they get the job done. Similar to the Canner, the cooling cycle draws unsterilised air back into the container.

Pros and cons of pressure sterilising mushroom substrate


  • Bulletproof/gold standard
  • Allows for high levels of Nitrogen/supplementation, resulting in higher yield
  • Relatively quick and easy to operate


  • High energy consumption/cost
  • Requires expensive equipment
  • The batch-focused nature of this process will dictates workflow. This can cause a bottleneck in production and capacity.
  • Requires an aseptic process from this point forward
  • Should not be left unattended during heating stages

Pressure Sterilisation tips

  • Steam, not water: Items to be sterilised must be in direct contact with the steam. If it is submerged in water it may not reach the required temperature.
  • Venting: All air in the container needs to be vented, and replaced by steam. If it is not, a pocket of compressed air (at a lower temperature) will not allow contact of the material with the steam and remain unsterilised. It will also lower the overall pressure within the system.
  • Pre germination: The most difficult thing to kill are the endospores of many bacteria and mould. However, if the prepared substrate is left to sit for 12-24 hours many of the spores will germinate making them easier to eliminate during sterilisation. Research has found that the increase in pressure aids in the germination of spores (during the heating stage), aiding in their elimination as the heat increases.
  • Air re-entry: If using a pressure cooker or canner, the sterilisation process forces air out of the container building positive pressure, but as it cools and the pressure drops creating negative pressure. The resulting vacuum may suck contaminated air back as the pressure equalises. This may then contaminate the outside of the bags. Or, in rare occasions, even make it inside the bag. Always treat the outside of the bag as contaminated. So, cleanse it (and your gloves after handling it) with isopropyl before opening.
  • Validators: There are things you can use to check that the sterilisation temperature has been reached. I use sterilisation indicator tape (it looks similar to masking tape) that will display black lines after the sterilisation temperature has been reached. I sometimes test my “cook” by putting these into the centre of the bag with a bit of cotton trailing. When I’m in the lab I will retrieve the tape and check.

Further research

2. Pasteurisation Method

Ideal for

  • Bulky low mass materials like straw
  • Good for faster-growing varieties like Oyster Mushrooms
  • Ideal for beginner to medium-level mushroom growers

How it works

  • Pasteurisation involves temperatures much lower than sterilisation, so is simple and easier to achieve. However, it may require a longer 'run' time, so a lot of energy to maintain.
  • The idea of pasteurisation is not to kill all the organisms within the substrate but allow certain beneficial ones to remain.


  • This method often uses a DIY approach to building the equipment necessary.
  • The ideal pasteurisation temperature is kept at around 70°C (158° F), requiring a thermostat (so electricity), to achieve.
  • Using gas or fire is possible but must be done with care, as it is prone to overheat.
  • There is a link below about using solar hot water for mushroom pasteurisation.

Time required

Depending on the thermal mass

  • 4-7+ hours to pasteurise
  • 2-12+ hours to cool down/drain

Equipment required

  • Large barrels, like a 44-gallon drum or plastic with a tight-fitting and vented lid.
  • An electric element and thermostat. This will need to be fitted in the inside bottom of the container. So seals and fittings will be required.
  • A suitable metal basket for holding the material and moving it into and out of the barrel & aid in draining.
  • Insulation to cover the container
  • Possibly rope and pulleys to lift and lower the basket into the barrel.

Pasteurisation, pros and cons


  • Relatively inexpensive setup, but may require DIY
  • Power consumption
  • Can handle large amounts of substrate.
  • Does not generally require aseptic conditions
  • Can be left unattended


  • Large dedicated containers
  • Long time to run and cool down
  • Best for substrate low in nitrogen/supplementation
  • The batch-focused nature of this dictates workflow so sets a bottleneck on production capacity

Tips for Pasteurisation

  • It may be possible to later convert the insulated barrel into a steam steriliser with the purchase of a steam generator. However, it will not reach full sterilisation temperature due to low pressure. But, comes close to most mushroom growers' needs.
  • Timing does not include the preparation of the substrate. For instance, straw should be shredded before pasteurisation.
  • The cool-down period is important and should be done in very clean conditions.
  • Inoculating warm substrate may result in contamination getting a head start.

Further research

3. Fermentation Method

Ideal for

  • Larger and bulky substrates like wood chips or straw or even smaller amounts like coffee grounds.
  • Ideal for beginners to advanced mushroom growers
  • Good for fast varieties growing varieties, like Oysters Mushrooms. Or to prep mulch for King Stropharia (Stropharia rugoso-annulata). King Stropharia will generally grow well in substrates without treatment as they may benefit from the higher levels of microbial action present.

How it works

  • Fermentation is perhaps one of the most natural ways to process substrate because it uses biological action. It is also a very low-tech technique, requiring only water. No energy is required.
  • The method involves submerging the substrate in water for at least a week. This allows the growth of anaerobic bacteria (who can only survive without oxygen) to proliferate. The endospores of these bacteria are naturally present, and waiting for air free conditions to germinate.
  • Over time, the organisms that require oxygen will die. The anaerobic bacteria will also scavenge other organisms and organic matter and consume it. This leaves the anaerobic bacteria the only thing left alive. They are later killed by draining the liquid and exposing them to air, leaving a fairly clean substrate.

Time required

  • Soaking time 1-3weeks
  • Airing/draining time 24+ hours

Equipment required.

  • Large drums or inflatable pool
  • Water

Fermentation method, pros & cons


  • Low cost
  • Can handle large amounts of substrate
  • Chunky dense substrate, mulch wood chips
  • Relatively dirty substrate materials
  • Does not require aseptic handling
  • Can be left unattended
  • The low-cost and low-tech nature of this means that


  • Getting rid of waste
  • Length of time it takes
  • Smelly

4. Microwave sterilisation

Ideal for

  • Small amounts of a substrate, like coffee grounds, Agar-Agar etc.
  • Ideal for Beginner to advanced mushroom growers

If you do not have a lab, and I would suspect this if you are interested in this method, a bonus is you could rig up a glove box type arrangement. Use a sheet of Perspex (with armholes and gloves attached) that is cut into a shape that fits when the door is open.

Time required

  • 5-12min sterilisation time
  • 10-120minute cooling time

Equipment required

  • High wattage microwave oven
  • Containers

Microwave sterilisation: pros and cons


  • Good for someone starting out.
  • Advanced mushroom growers looking for a fast and simple way to sterilise small batches
  • Fast and convenient
  • not a lot of preparation required


  • Fire risk!
  • Only good for a small quantity of substrate
  • Will Require aseptic handling and conditions
  • can not be left unattended


  • Only small batches can be processed - less than 1kg total.
  • Items with a larger thermal mass will take longer and risk a fire.
  • The substrate and materials require a high moisture content to produce high-temperature steam. Obviously, no metal objects should be placed inside.
  • Use a steam steriliser, sold for cleaning babys bottles (seen in the photo below)
  • Use a Pyrex bowl or only plastic bags that can handle high heat. Grow bags are okay.
  • Use a sterilisation indicator to experiment with how long this will be based on the mass you have, and the wattage of your microwave.
  • Because of its utility and low cost, a dedicated microwave is a good addition to any lab. See the links below for further reading.

further research


Worth repeating: This can be a fire risk, do not perform unattended

microwave steam steriliser

5. Hydrated lime (aka calcium hydroxide)

Ideal for

  • Wood pellets or similar dry materials that will soak up all liquid
  • Bulky material that will partially soak up the liquid, like straw and mulch
  • Ideal for beginners to advanced mushroom growers

This is sometimes called cold pasteurisation because many people think that the high pH obtained kills potential contaminants… but it doesn’t. If it did it would also kill the mushroom mycelium.

Hydrated Lime, also known as: Calcium Hydroxide or CaH2O2 is a fairly organic compound made by superheating naturally occurring lime so it bonds with some extra atoms of Hydrogen and Oxygen.

How it works

  • The addition of Hydrated Lime pushes up the pH to make the mixture that is very alkali (caustic). This makes the mixture too “hostile” for spores of bacteria or other fungi to germinate.
  • The mycelium of the mushroom, which is already growing is less affected. It continues to colonise the substrate relatively unimpeded.
  • Over time however, the mushroom will buffer the substrate closer to neutral (pH7) in which contaminants may germinate. If they do, we’re hoping that the mushroom culture has established and will use their wonderful immune system to deal with an outbreak.
  • If mould is already growing in the spawn (or substrate) it will continue to grow and possibly colonise the bag instead. Much like it would if the bag had been sterilised.

How to

  • About .5g of Hydrated Lime per litre of water. It Needs to be mixed or it will settle. Much of it will be suspended not dissolved in the water.
  • Adding too much hydrated lime is less of a problem than not adding enough. Adding more will not make the pH go above 14 as the water will be fully saturated with the Lime. Too little may result in not reaching the desired PH level.

When Soaking

  • If using straw or woody substrate it may require soaking for up to 12 hours.

When Rehydrating

  • Using Wood pellets, soaking is not required. Just use a sufficient amount of lime saturated water to hydrate the batch to field capacity.

Time required

  • If rehydrating: 15-20 minutes (the length of time required to mix and hydrate the substrate.
  • If Soaking: 12+ hours

Equipment required.

  • Hydrated Lime: Make sure it is Calcium Hydroxide (with low magnesium). There are similar lime concoctions available that will not work.
  • PH test strips

A mixing or soaking container, depending on the method:

  • Soaking, A large drum. For bigger loads, something like an inflatable pool.
  • Mixing: For practicalities, this is often a cement mixer.

Hydrated Lime process, pros & cons


  • Relatively cheap, with no energy required
  • A fast process if the substrate soaks up all the liquid (ideal for the hydrating of a pelletised substrate.
  • Easy for large and small production.
  • Does not require aseptic condition or handling
  • Can be left unattended


  • Only good for faster-growing varieties
  • less supplementation (no more than 30% by dry weight) should be added possibly resulting in a lower yield.
  • If soaking and not absorbed, need to get rid of waste


  • If adding a nitrogen-based supplement, like Soy hull pellets, add these to the mix first and fully hydrate. If there’s going to be a contamination problem it will start here.
  • Always use cold water for this method. Warm water will encourage growth of contaminants.
  • If you smell ammonia during the mixing. It’s most likely that the spawn has been contaminated with a bacteria or yeasts that excrete urea. A chemical reaction will then occur creating ammonia. What’s going on can be expressed in this chemical equation. The 2NH3 is the offending smell.
    CaH2O2 + CH4N2O → CaCO3 + 2NH3
    Note: the ammonia will have no effect on the bacteria or mould present.
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