Exercise 6
Fungi and Mushroom
Introduction
The market for mushrooms continues to grow due to interest in their culinary, nutritional, and health benefits. Mushrooms contain many essential amino acids; white button mushrooms, for example, contain more protein than kidney beans. Shiitake mushrooms are less nutritious, but are still a good source of protein (Royse and Schisler, 1980). As a group, mushrooms also contain some unsaturated fatty acids, provide several of the B vitamins, and vitamin D. Some even contain significant vitamin C, as well as the minerals potassium, phosphorus, calcium, and magnesium (Park, 2001). However, as fungi, mushrooms have life cycles very different from those of green plants. Mushrooms do not contain chlorophyll and therefore depend on other plant material (the "substrate") for their food. The part of the organism that we see and call a mushroom is really just the fruiting body. Unseen is the mycelium, tiny threads that grow throughout the substrate and collect nutrients by breaking down the organic material. This is the main body of the mushroom. Generally, each mushroom species prefers a particular growing medium, although some species can grow on a wide range of materials. The choice of species to be raised depends both on the growth media available and on market considerations. Oyster mushrooms, which grow on many substrates, are easiest for a beginner. Shiitake mushrooms already have earned considerable consumer demand. Small-scale mushroom production represents an opportunity for farmers interested in an additional enterprise and is a specialty option for farmers without much land.
The goal of this exercise is to start with one mushroom fruiting body and turn it into a bunch of new fruiting bodies. There are two mechanisms by which mushrooms can reproduce -- by spores or by propagation of tissue extracted from a fruiting body. We will use the latter technique, which is simpler and which offers a better chance of success for the beginning cultivator.
There are three distinct phases (fig 1) through which your culture will pass:
1. Sterile culture on an agar medium
2. Sterile culture on grain, also known as "grain spawn"
3. Fruiting on a pasteurized substrate
Objectives
Students are able to produce their own mushroom cultures starting with a sterile piece of mushroom tissue and transfer it onto a series of media and, finally, induce to fruiting bodies.
Figure1. An overall scheme of mushroom cultivation
1. Sterile culture on an agar medium
Materials
1. Potato Dextrose Agar (PDA) ingredients (see experiment 2.3)
2. Sterile Petri dishes
3. Scalpels
4. Alcohol lamps
5. Fresh, clean mushroom fruiting bodies (oyster mushrooms are often recommended for beginning cultivators)
Procedure
1. Prepare PDA (see experiment 2.3). Pour into Petri dishes. Leave at room temperature to solidify and dry overnight.
2. In a sterile flow hood, light an alcohol lamb. Sterilize the scalpel blade thoroughly in the flame, then place it on a stand.
3. Tear or break off a section of the mushroom (do not cut with a knife, as the blade will introduce contaminants from the outer surface).
4. Starting with the tip of the scalpel, cut a small square shape, approximately 0.5 cm x 0.5 cm, from the clean inner tissue preferably where the mushroom cap and stalk are connected and gently tear the tissue free.
5. Place the tissue in the middle of the PDA Petri dish. Date and label the content using a permanent marker.
6. Place the newly inoculated dishes in a low light location at room temperature. In a few days to a week, the inoculated tissue will become fuzzy as the mycelium begins to grow. After several weeks, mycelium will completely cover the agar surface.
2. Sterile spawn culture on grain
Materials
1. Clear 12 oz. soda water bottles
2. Rolled cotton
3. Grain (wheat, rye, millet or sorghum)
4. Aluminum foil
5. Inoculation loops
Procedure
1. Prepare grain. Add 1/3 cup of sorghum or other grain and 1/4 cup water into a 12 oz. bottle. Plug the bottle with rolled cotton. Cover the plug with aluminum foil.
2. Place the bottles with grain into an autoclave. Cook at 121 oC, 15 psi for seven minutes. Let the autoclave cool and pressure down. Use oven mitts to shake the hot bottles so that wet and dry grain is mixed. Place the containers back in the pressure cooker and sterilize by cooking at 15 psi for 45 minutes. Remove the bottles from the autoclave and shake once again to mix the grain. Leave the bottles to cool down.
3. Remove the lid of the PDA plate and the cotton plug of the bottle, use sterile inoculation loop to cut a small piece of mycelium from the agar. Place it just inside the bottle and close the lids. Tap the bottle against the heel of the hand until the mycelium is positioned in the grain.
4. Once the bottles have been inoculated, date and label their contents using a permanent marker. The mycelium of mushrooms like oyster mushrooms and Agaricus grows well at room temperature. Periodically shake the container to speed colonization and to prevent the grain from clumping into a thick mass. Once a grain culture is fully colonized, it can be stored in a refrigerator to maintain its viability.
3. Fruiting on a pasteurized substrate
Materials
1. Rice straw
2. Wheat or rice bran (or grains such as rye, sunflower seed in the hull, or millet)
3. Calcium carbonate
4. Clear plastic bags (8”x11”)
5. Plastic bottle necks
6. Sterile rolled cotton
Procedure
1. Make straw based mushroom growing substrate. Cut 2 kg of straw into 1-3 inch long pieces. Add 2 cups wheat or rice bran (or grains such as rye, sunflower seed in the hull, or millet) and 1 teaspoon calcium carbonate to the straw and place the mixture into a cloth bag. Optionally, pre-soak the straw for 3 days prior to pasteurization.
2. Completely submerge the bag in hot water (65°C) for approximately 1 hr to provide pasteurization.
3. Take the container of straw out of the hot water and place it on a clean, disinfected surface to drain and cool to room temperature. This will take approximately 1-2 hours.
4. Tightly pack the cooled straw into a clear plastic bag about ¾ of the bag. Ensemble a plastic bottle neck onto the opening of the bag. Plug it with the rolled cotton.
5. Inoculate the straw substrate with the spawn. Shake the spawn bottle well before opening. Unplug the straw bag. Pour approximately 1 teaspoon of grain into the straw bag. Plug it with the rolled cotton.
6. Place the spawned bags in a humid and dark place with good circulation. In approximately two weeks the substrate should be completely colonized with creamy-white mycelium.
7. Open the bag and remove the bottle neck, wart like growths will appear in approximately one week. These will quickly grow into mushrooms.
References
แบบฝึกหัดที่ 6เชื้อราและเห็ดแนะนำThe market for mushrooms continues to grow due to interest in their culinary, nutritional, and health benefits. Mushrooms contain many essential amino acids; white button mushrooms, for example, contain more protein than kidney beans. Shiitake mushrooms are less nutritious, but are still a good source of protein (Royse and Schisler, 1980). As a group, mushrooms also contain some unsaturated fatty acids, provide several of the B vitamins, and vitamin D. Some even contain significant vitamin C, as well as the minerals potassium, phosphorus, calcium, and magnesium (Park, 2001). However, as fungi, mushrooms have life cycles very different from those of green plants. Mushrooms do not contain chlorophyll and therefore depend on other plant material (the "substrate") for their food. The part of the organism that we see and call a mushroom is really just the fruiting body. Unseen is the mycelium, tiny threads that grow throughout the substrate and collect nutrients by breaking down the organic material. This is the main body of the mushroom. Generally, each mushroom species prefers a particular growing medium, although some species can grow on a wide range of materials. The choice of species to be raised depends both on the growth media available and on market considerations. Oyster mushrooms, which grow on many substrates, are easiest for a beginner. Shiitake mushrooms already have earned considerable consumer demand. Small-scale mushroom production represents an opportunity for farmers interested in an additional enterprise and is a specialty option for farmers without much land.The goal of this exercise is to start with one mushroom fruiting body and turn it into a bunch of new fruiting bodies. There are two mechanisms by which mushrooms can reproduce -- by spores or by propagation of tissue extracted from a fruiting body. We will use the latter technique, which is simpler and which offers a better chance of success for the beginning cultivator.
There are three distinct phases (fig 1) through which your culture will pass:
1. Sterile culture on an agar medium
2. Sterile culture on grain, also known as "grain spawn"
3. Fruiting on a pasteurized substrate
Objectives
Students are able to produce their own mushroom cultures starting with a sterile piece of mushroom tissue and transfer it onto a series of media and, finally, induce to fruiting bodies.
Figure1. An overall scheme of mushroom cultivation
1. Sterile culture on an agar medium
Materials
1. Potato Dextrose Agar (PDA) ingredients (see experiment 2.3)
2. Sterile Petri dishes
3. Scalpels
4. Alcohol lamps
5. Fresh, clean mushroom fruiting bodies (oyster mushrooms are often recommended for beginning cultivators)
Procedure
1. Prepare PDA (see experiment 2.3). Pour into Petri dishes. Leave at room temperature to solidify and dry overnight.
2. In a sterile flow hood, light an alcohol lamb. Sterilize the scalpel blade thoroughly in the flame, then place it on a stand.
3. Tear or break off a section of the mushroom (do not cut with a knife, as the blade will introduce contaminants from the outer surface).
4. Starting with the tip of the scalpel, cut a small square shape, approximately 0.5 cm x 0.5 cm, from the clean inner tissue preferably where the mushroom cap and stalk are connected and gently tear the tissue free.
5. Place the tissue in the middle of the PDA Petri dish. Date and label the content using a permanent marker.
6. Place the newly inoculated dishes in a low light location at room temperature. In a few days to a week, the inoculated tissue will become fuzzy as the mycelium begins to grow. After several weeks, mycelium will completely cover the agar surface.
2. Sterile spawn culture on grain
Materials
1. Clear 12 oz. soda water bottles
2. Rolled cotton
3. Grain (wheat, rye, millet or sorghum)
4. Aluminum foil
5. Inoculation loops
Procedure
1. Prepare grain. Add 1/3 cup of sorghum or other grain and 1/4 cup water into a 12 oz. bottle. Plug the bottle with rolled cotton. Cover the plug with aluminum foil.
2. Place the bottles with grain into an autoclave. Cook at 121 oC, 15 psi for seven minutes. Let the autoclave cool and pressure down. Use oven mitts to shake the hot bottles so that wet and dry grain is mixed. Place the containers back in the pressure cooker and sterilize by cooking at 15 psi for 45 minutes. Remove the bottles from the autoclave and shake once again to mix the grain. Leave the bottles to cool down.
3. Remove the lid of the PDA plate and the cotton plug of the bottle, use sterile inoculation loop to cut a small piece of mycelium from the agar. Place it just inside the bottle and close the lids. Tap the bottle against the heel of the hand until the mycelium is positioned in the grain.
4. Once the bottles have been inoculated, date and label their contents using a permanent marker. The mycelium of mushrooms like oyster mushrooms and Agaricus grows well at room temperature. Periodically shake the container to speed colonization and to prevent the grain from clumping into a thick mass. Once a grain culture is fully colonized, it can be stored in a refrigerator to maintain its viability.
3. Fruiting on a pasteurized substrate
Materials
1. Rice straw
2. Wheat or rice bran (or grains such as rye, sunflower seed in the hull, or millet)
3. Calcium carbonate
4. Clear plastic bags (8”x11”)
5. Plastic bottle necks
6. Sterile rolled cotton
Procedure
1. Make straw based mushroom growing substrate. Cut 2 kg of straw into 1-3 inch long pieces. Add 2 cups wheat or rice bran (or grains such as rye, sunflower seed in the hull, or millet) and 1 teaspoon calcium carbonate to the straw and place the mixture into a cloth bag. Optionally, pre-soak the straw for 3 days prior to pasteurization.
2. Completely submerge the bag in hot water (65°C) for approximately 1 hr to provide pasteurization.
3. Take the container of straw out of the hot water and place it on a clean, disinfected surface to drain and cool to room temperature. This will take approximately 1-2 hours.
4. Tightly pack the cooled straw into a clear plastic bag about ¾ of the bag. Ensemble a plastic bottle neck onto the opening of the bag. Plug it with the rolled cotton.
5. Inoculate the straw substrate with the spawn. Shake the spawn bottle well before opening. Unplug the straw bag. Pour approximately 1 teaspoon of grain into the straw bag. Plug it with the rolled cotton.
6. Place the spawned bags in a humid and dark place with good circulation. In approximately two weeks the substrate should be completely colonized with creamy-white mycelium.
7. Open the bag and remove the bottle neck, wart like growths will appear in approximately one week. These will quickly grow into mushrooms.
References
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