Our resident mycologist, John Robinson, shows us how to obtain spores from mushrooms to help them multiply into their millions! Mmmmmmmm!

Spores can be obtained from mushrooms easily. The problem is that they come in a form that makes them pretty difficult to use – they are very small and must be kept free of contaminants for successful cultivation. One solution is to prepare a suspension of spores in sterile water, which can be easily stored and used for cultivation or microscopy.

How to Take a “Spore Print”

Spores are usually released from the underside of the mushroom caps. To collect these spores, all we need to do is to place a freshly cut cap onto a clean piece of paper (take from the centre of a new pack) or some clean tinfoil (straight from part of a roll that hasn’t been exposed to the environment). When carrying out the following steps it is always best to wear latex gloves and use alcohol hand rub.

It is possible to take prints from mushrooms bought in the supermarket but these are often a bit dry, so get them as fresh as possible. It’s much better to take them from your own cultivated mushrooms.

Once you have your mushroom, simply hold the cap from above by its edges and cut the stem where it joins the cap. Place the cap flat on the paper/tinfoil and place an upturned bowl over it to reduce air movement. Leave for 12-24 hours and you should see a fine dust on the foil – these are millions of tiny mushroom spores! They can be any color from black to white, depending on species. Carefully fold the paper, taking care not to touch the spores, and place it in a plastic bag for storage in a cool dark place (a fridge is ideal). Spores collected in this manner can last for many years.

Taking spore prints from golden oyster mushrooms.

Constructing a “Glove Box”

We are surrounded by microorganisms – they are present on our skin, our clothes, and floating through the air. If we can eliminate these organisms from a small area, we can create a workspace for the preparation of sterile samples and agar plates. A glove box is a semi-sterile environment with little air movement that can be easily cleaned. A simple home version can be made using a transparent plastic storage box and lid with two holes cut into the front. The cultivator can access the work area via the holes. A long pair of rubber gloves can be glued in place around the holes but this isn’t essential. A more advanced version has a HEPA air-purifier unit and fan fixed into a hole in the back. The HEPA filter unit removes particles and contaminants from the air and blows cleaned air over the work area (see diagram below). More information can be found at www.micro-science.co.uk.

Once you have your glove box and spores you can move on to the next step: sterilizing a container.

Choosing a Container

Caution: Remember your safety is your responsibility. Be careful handling items straight from the microwave or pressure cooker as they are very hot! Also, syringes and needles should be disposed of carefully (e.g. needles into a sharps container) and discarded appropriately after use. Remember that alcohol is flammable. Do not work near flames or fire hazards.

A simple container can be made using a glass flask loosely plugged with cotton wool. Hydrophobic cotton wool is best, as this does not become waterlogged during sterilization. A narrow-necked vessel is best as it reduces the risk of contamination entering later. Shown below is a 3.5 oz (100mL) Erlenmeyer flask:

A slightly more advanced vessel is to the left– it consists of an Azlon jar with two half-inch (13mm) holes drilled through the lid. A butyl rubber injector port can then be fixed in one hole using plastic glue and the other hole plugged with hydrophobic cotton wool.

Sterilizing Your Container

A small amount of water is placed in the bottom (approximately 1.7 oz in this 3.5 oz Erlenmeyer flask, approximately 3-7 oz in a larger jar) and microwaved for two to three minutes. Allow the container to cool and microwave a second time. Alternatively, pressure-cook the container for 15-20 minutes.

Carefully remove the (hot!) container and check that the cotton wool is still in place. Screw the lid on tightly if using an Azlon jar. Cover with tinfoil and allow everything to cool down to room temperature before proceeding (at least a couple of hours). Once cool, check that the lid is screwed tightly on again if using a jar.

Making Sterile Spore Syringes

Checklist:

• Spore print
• 70% Alcohol spray or 10% bleach solution
• Alcohol hand rub
• Flask or jar of sterile water (with cotton wool stopper)
• Sterile inoculation loops*
• Sterile syringes and needles
• Pressure cooker or microwave

*It is possible to use a metal loop or scalpel and sterilize it using a butane lighter or methylated spirit burner. I would recommend using sterile plastic loops as this eliminates the need for a flame in the work area, which is a potential fire hazard, especially if alcohol is used.

Find a still, quiet location for your glove box where you will be able to work comfortably (such as a desk). Spray and wipe down the inside with disinfectant and place all the items you are going to work with inside. Spray and wipe down again. Have a couple of practice runs to make sure you have everything in place and are confident. Once you begin you will need to work quickly to reduce the risk of airborne contamination. For best results wear latex gloves, and use alcohol hand rub.

Step 1: Take your spore print out of the bag so it’s ready to use. Remove the cotton wool bung and hold it carefully (don’t allow it to touch anything) as you scrape the spores using a sterile instrument in through the hole in the lid. Replace the cotton wool bung.

Step 2: If you are using a glass container, simply remove the cotton wool and withdraw the spore suspension using a sterile syringe. If using the Azlon jar, swab the rubber injector port with an alcohol wipe, incline the jar and use a sterile syringe to withdraw spore suspension. This process can be repeated to get a number of syringefulls.

Step 3: Spores can often be seen floating in the syringes. However, if you don’t see them, don’t worry – there may still be lots present. Spore syringes can be stored for months, maybe years, in the fridge. However, they do become less viable and germinate less readily the longer they are left.

Final Thought

Once you have mastered the art of making spore syringes you can become truly self-sufficient. It can require some trial and error at first, but is well worth it in the long run. Happy mushroom farming!

Sounds easy so far! Now what do we do with all those viable spores? Stay tuned for more mushroom-growing lore and next-steps from our favourite fungi-guru.