Quickly, push a small amount of cotton wool through the lid's breathing hole and attach to the jar. Using the scalpel, transfer the wedges to the sterile grain. Remove the aluminium foil and lid of the grain jar. Sterilise the scalpel blade, remove the Petri dish lid and cut 2 wedges from the centre of the dish. Choose only the most healthy cultures for the inoculations. Once fully colonised (mycelium nearing the edges), it is time to transfer the agar to the sterilised grain. Colonisation should take approximately 8 to 10 days.ĭuring this time, remove any Petri dishes that appears to be contaminated with other moulds. Incubate for pleurotus ostreatus at 24☌ (75☏) and for pleurotus pulmonarius (summer) 24☌ to 30☌ (75☏ to 85☏). Repeat the process, making sure to sterilise the scalpel before each transfer. You may wish to tape the lid (with a clean breathable tape) to reduce the chance of contamination. Place the square of tissue centrally into the agar and cover with the Petri dish lid. With the scalpel, carefully cut a small square from the newly exposed mushroom tissue. Lift the lid of the Petri dish and cool the scalpel blade by placing it centrally into your agar. Sterilise the scalpel blade by holding it within the alcohol burner's flame. Place the mushroom (outside down) on to a clean surface, making sure you keep the inside tissue from touching anything. Taking the mushroom by its base, carefully spit it in two. A laminar flow bench and hepa filter would reduce contamination during this stage, but it is possible (with a higher contamination rate) to succeed without one. Spray the clean room walls, benches and floors with 5% bleach solution (as before wear clean clothes, wash your hands etc). Allow the Petri dishes to cool completely. Move your Petri dishes, mushroom tissue and other equipment to the clean room. Wrap in aluminium foil and pressure cook with your grain (or for at least 30 minutes at 15 psi). Pour a thin layer into your Petri dishes and cover with lids. As it begins to boil, continue to stir for a minute and then remove from the heat. Begin to heat and stir until the agar is completely dissolved. Measure out 5.75 grams of nutrient agar powder to 1 cup of clean water (ample for 5 or more Petri dishes). For pleurotus ostreatus incubate at 24☌ (75☏) and for pleurotus pulmonarius (summer) 24☌ to 30☌ (75☏ to 85☏).Īnother method to inoculate your grain, is by first propagating the mushroom tissue on Agar (or cloning). Place on a shaded shelf within the clean room to incubate. Finally, shake the jar vigorously to disperse the grain spawn throughout the jar. Transfer 1-2 desert spoons of the spawn into your 1 litre jar of sterile grain. Open your spawn bag (or jar) and taking your sterile utensil of preference, break up the grains ready to transfer. Remove the aluminium foil and lid of your jar. A face mask and hair cap will also help reduce contamination (we are very dirty creatures). Clean your hands with antibacterial soap or wear sterile gloves. Spray down the clean room walls with a 1:20 ratio (5%) of bleach to water (It is suggested that a HEPA filter be employed to clean the air, however, this instructable is low tech). Make sure you have showered and are wearing clean clothes. You can purchase your initial grain spawn online and have it delivered. One of the most straight forward and successful methods, is grain spawn transfer. There are a number of methods to inoculate the sterilised grain. This instructable uses a low tech approach and will give you alternative methods for minimising air contaminants. Most mushroom clean rooms have a HEPA filter installed to provide clean oxygenated air. The room also requires a work bench and storage shelves for incubating your jars of spawn. For an opening, you can cut a suitably sized slit, attaching duct tape tabs with stick on Velcro acting as fasteners. The room should be able to be easily wiped down with a bleach solution. Finally, tape a plastic sheet over the top of the room to form its ceiling. Run sealant between the base of the walls and the floor. Screw the four panels together using sealant along the joins. Cover these panels with plastic sheeting (use staples to attach). Using thin lengths of wood, nail these together to make four wall panels. This can be achieved by constructing a simple clean room. Air is full of impurities and so it is important to reduce the level of containments where possible. To perform the grain inoculations, you require a sterile environment.
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