How to produce sterile substrate

How to produce sterile substrate, using

Mushroombox Mycobags.

This document is not the definitive method for producing sterile material, but can serve as a basis from which you can develop your own procedures. No responsibility is taken for failure. Please ensure that you assess any risks involved and are competent to carry out this procedure before attempting. You follow this procedure at your own risk!

What you need:

Mushroombox mycobag

Pressure cooker

Substrate (eg oak sawdust, straw etc).


  1. Place substrate in the bag. Typically you would fill the bag between 1/3 and ½ full – no more. For standard sized pressure cookers, you may need to use less than this, in order to fit inside the pressure cooker. Place the bag in a container so that there is no danger of it tipping over (ideally in a sink or basin). Pour on boiling water until the substrate is completely immersed and soaked-through. Some substrates are highly-absorbent – in which case you'll need an excess of boiling water to compensate.

  2. Leave the substrate to soak for a couple of hours, to make sure it's fully hydrated.

  3. Pour off the excess water, taking appropriate precautions against scalding. Make sure there is no residual water whatsoever – this is critical,and one of the commonest mistakes. If you are using the same substrate every time, you can establish through trial and error, the exact amount of water to rehydrate the substrate without producing any excess.

  4. Fold the top of the bag over a couple of times, expelling ALL of the air inside the bag in the process. This is very important – make sure there is no air left in the bag. You can tape the folded section to the body of the bag with masking tape or autoclave tape to keep it from opening after sterilisation

  5. Wrap the bag in a damp tea-towel (NB use an old tea towel, as it will more than likely suffer damage during pressure-cooking)

  6. Add about 25mm depth of water to the pressure cooker, and place the tea-towel wrapped bag of substrate inside the pressure-cooker, ensuring there is adequate clearance for the lid to close properly, without impeding the operation of the safety valves.

  7. Place the pressure-cooker onto the hob, and when the valve starts hissing reduce the heat to a level that maintains a low but steady emission of steam from the pressure-cooker.

  8. Cook for a minimum of 45minutes. For larger volumes inside the bag, you may wish to increase the cooking time to a maximum of 2hours. You need to provide sufficient time for sterilisation temperatures to be reached in the centre of the bag, but not so much that you render the substrate to a paste!

  9. Turn off the heat and remove from hob to cool fully to room temperature (this will be several hours – so often best to leave overnight)

  10. In a clean environment (ideally a sterile air flow from a laminar hepa filter), remove the lid, and remove the bag of substrate. Open the top of the bag for as short a time as possible, add the mushroom spawn and immediately seal the top with a heatsealer (or failing that, fold it over and tape it closed.

  11. Incubate the substrate/spawn at the desired temperature until full colonisation occurs.

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