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Mushroom cultivation is a rewarding hobby that allows you to grow your own delicious and nutritious mushrooms at home. One essential tool for mushroom cultivation is agar, a solidifying agent made from seaweed that is used to create a solid surface for the growth of a monoculture (a culture containing only one type of organism). Agar mushroom growing is an advanced topic.
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In this article, we will introduce you to the basics of agar mushroom growing. We will be covering using agar to cultivate monocultures and start new mushroom-growing projects, as well as how to transfer your cultures to new agar plates. Whether you are a beginner looking to get started in mushroom cultivation or an experienced grower looking to expand your knowledge, this post will provide you with the information you need to get started.
If you’re new to mushroom growing, we recommend starting with this post here.
What Is Agar?
Agar is made from a type of seaweed called Gelidium, which is dried and ground into a powder. When mixed with water, agar forms a gel-like substance that is solid at room temperature but melts when heated.
Agar has a number of useful properties that make it an ideal choice for use in mushroom growing and in various other laboratory applications. For example, it is not digested by most microorganisms, making it a suitable substrate for the growth of a wide range of organisms. It is fairly resistant to contamination, which is useful for the cultivation of pure cultures.
In addition to its use in the cultivation of microorganisms, agar has a number of other applications. It is used in the food industry as a thickening agent and as a vegetarian alternative to gelatin. It is also used in cosmetics and pharmaceuticals as a stabilizer and gelling agent.
Overall, agar is a versatile and widely-used substance that has many applications in the fields of science, industry, and beyond. However, we will stick to the mushroom cultivation part, as that is our area of expertise!
Why Use Agar in Mushroom Cultivation?
Agar is a solidifying agent that is essential for the cultivation of pure, uncontaminated monoculture mushroom cultures. It is recommended to germinate spores on agar before using them for cultivation, as spores can sometimes be accompanied by contaminants such as yeast and mold. By cultivating mycelium on agar plates, it is easier to identify any potential contaminants and make a clean transfer to a new agar plate.
Read more about agar on Wikipedia.
In addition to germinating spores, agar plates are also useful for creating fungal clones. Instead of germinating spores, a clean fragment of the mushroom fruiting body can be placed on agar to grow. This allows the cultivator to isolate any potential contaminants that may be present.
How To Use Agar
There are various recipes for agar mixes that can be used in mushroom cultivation, such as MEA (malt extract agar), PDA (potato dextrose agar), and MYA (malt yeast agar). There are even methods that utilize dog food like DFA (dog food agar). The basic principle for all these methods is the same, add a food source to agar in order to create a nutrient-rich media for mushroom cultures. Regardless of the specific recipe, all agar mixes involve the creation of a nutrient-rich substrate for the growth of mushroom cultures. For this tutorial let’s stick with MEA.
What Will You Need
- 20 grams agar
- 20 grams of light malt extract
- 1000 ml water
- Petri dishes
- Media bottle or jar with a hole in the lid
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How To Prepare Agar Petri Dishes
Step 1: Weigh the dry ingredients
Using a precision scale, weigh the light malt extract and agar powder. Add these dry ingredients to a heat-proof jar.
Step 2: Measure out boiling water
Use a kitchen scale to measure out hot water (in grams). Add it to the jar containing the dry ingredients and mix well until dissolved.
Step 3: Sterilize the agar
Pour the dissolved agar and light malt extract mixture into a bottle for sterilizing. Secure the lid on the bottle.
Sterilize the agar mixture for 20 minutes at 15 PSI in a pressure cooker. Allow the pressure cooker to fully depressurize, or wait at least 1 hour, before opening it.
Step 4: Cool the agar
Place the bottle of hot agar mixture in a warm water bath. Use a thermometer to monitor the temperature. The agar mixture should reach the same temperature as the water bath.
To reduce condensation, cool the agar to 55 degrees Celsius before pouring the plates.
Step 5: Pour the agar
Prepare a sterile workspace for pouring the plates. It may be under a flow hood or in the glove box, just keep it as sterile as possible.
Start by pouring one plate at a time and stacking them on top of one another. Make sure to work your way up without disturbing the already-poured plates. Pour approximately 20 mL of agar into each Petri dish. With this recipe, you should have 40 agar plates. If you decide it’s too much just split everything in half.
Step 6: Seal and store the plates
Once the agar has cooled and set, you can seal the plates using parafilm or plastic wrap. Any unused plates can be stored in the refrigerator until needed.
How To Inoculate Agar Plates
To minimize the risk of contamination when working with open agar plates, it is recommended to use a flow hood or Still Air Box (SAB). When using these tools, follow these steps to inoculate your agar plates:
1. Remove the agar plates from the sealed bag within your SAB or flow hood
2. Stack the plates up and work with one open plate at a time.
3. Depending on the type of inoculum you are using, follow these steps:
- Spore syringes: Place a tiny drop (<1cc) on the center of the plate. Use a sterile scalpel, or inoculation loop, or simply move the plate around to spread the liquid around.
- Spore print: Using a sterile scalpel or inoculation loop, scrape a small amount of spores onto the agar surface.
- Liquid culture: Use the same method as with spore syringes.
- Cloning: Gently pull apart the mushroom (do not cut) within your flow hood/SAB. Using a scalpel, cut a tiny grain-sized piece of the inner tissue of the cap or stem. The tissue should stick to the scalpel. Push the tissue onto the agar, allowing the scalpel to cut through the tissue and into the agar. This should cause the tissue to stick to the agar surface.
4. Once you have inoculated the agar plate, place the lid on it and use the included parafilm to seal the plate.
Understanding and Preventing Contamination in Agar Mushroom Growing
In agar culturing, contamination refers to the presence of any microorganism (such as mold, bacteria, yeasts, or fungi) that is not the intended organism being cultivated. When working with agar or other nutritious media, it is essential to consider all potential sources of contamination and take steps to minimize the risk. While it is not always possible to completely eliminate contamination, most sources can be controlled to acceptable levels through careful planning and following proper techniques.
How To Prevent Contamination
There are several key factors to consider when attempting to prevent contamination in agar culturing. These include:
Using reliable and clean inoculum sources, such as spore syringes, spore prints, or liquid cultures from trusted vendors. Agar mushroom growing is no exception to the level of care you need to put into growing mushrooms.
Understanding that clone specimens, whether cultivated or wild, may contain contaminants and may require multiple transfers to achieve a clean culture.
Read more about mushroom contamination on our blog post here.
Sterilizing all tools, such as scalpels and inoculation loops, before use through methods such as pressure cooking or flame sterilization.
Working in a controlled environment, such as a flow hood or Still Air Box (SAB), to minimize the introduction of contaminants from the air.
Practicing good personal hygiene, including wearing nylon gloves that can be sanitized with alcohol before working with agar plates.
Ensuring that the Parafilm tape used to seal the agar plates covers the entire edge of the plate with no gaps or holes, as even small openings can allow contaminants to enter.
How To Spot Contamination – Agar Mushroom Growing
When the mycelium (the white vegetative growth of fungi) begins to grow on the agar plate, it will spread out across the surface and appear as a white fuzziness emanating from the area of inoculation. Other microorganisms, such as molds, bacteria, and yeasts, may also appear on the plate in small spots but will spread more quickly than the mycelium.
Yeasts and bacteria will often have a white or yellowish, milky appearance. On the other hand, molds will resemble mycelium in appearance but will spread more rapidly and may turn green or black. Avoid opening agar plates that have a dark green, white, or black powdery appearance around clean agar plates, as this could be a sign of contamination.
Agar Plate Incubation – Mushroom Growing
In general with agar mushroom growing, agar plates can incubate at room temperature for most species. However, some species may require a higher temperature to encourage germination. In these cases, an incubation chamber can be used to maintain the optimal temperature. It is important to be aware of the specific growth parameters of the species you are working with and ensure that you are providing the proper conditions for optimal growth.
The process of germinating spores on agar can take up to 6-8 weeks to see any growth. This time is for spore germination, hyphae growth, and fusion into mycelium. Once noticeable growth begins, you may start to see signs of sectoring, which indicates the presence of varying genetics due to different spores containing different genetic characteristics. If desired, you can isolate certain sections of the mycelium based on vigorous growth.
Transferring Agar to Agar
Once the mycelium has grown to cover approximately 80-90% of the agar plate, you can choose to transfer the culture to additional agar plates. This process, known as “transferring,” allows you to create new cultures while leaving behind any contaminants that may be present. If your agar plate has become contaminated, you can also transfer uncontaminated sections to a new plate in order to obtain a clean culture. It may be necessary to repeat the transferring process multiple times in order to achieve a completely clean culture.
When transferring a culture from one agar plate to another, it is important to choose the best candidate for transfer. This may be the section of the plate with the least contamination or the most vigorous growth. To transfer a culture, follow these steps:
- Working within a flow hood or SAB, open the colonized agar plate.
- Remove the parafilm tape from the clean agar plate, leaving the lid in place.
- Flame sterilize a scalpel until it is red hot.
- Using the sterilized scalpel, cut a small 1×1 cm square of mycelial growth from the leading edge of the colonized agar plate.
- Using the scalpel, pick up the square of mycelium and transfer it to the middle of the clean agar plate. Close the lid of the clean agar plate.
- Seal the agar plate using parafilm, plastic wrap, or even blue painter’s tape. This helps to prevent contamination and maintain the optimal growth conditions for the transferred culture.
Once a clean culture is on an agar plate, it can be stored in a sealed container in the refrigerator to slow down its growth. This allows the culture to be used at a later date without the need for frequent transfers. It is important to ensure that the container is properly sealed in order to prevent contamination and maintain the viability of the culture.
The Bottom Line
Whether you are a beginner or an experienced mushroom grower, agar culturing is a valuable technique in mushroom cultivation. Agar is a solidifying agent that forms a gel-like substance when mixed with water and is resistant to contamination, making it an ideal medium for pure cultures. To start a culture using agar, you will need to sterilize your equipment and ingredients and prepare an agar medium by mixing agar with water and any additional nutrients required by the organism.
Once the agar has solidified, you can transfer a small piece of mushroom to the agar using a sterilized tool, such as a scalpel or a toothpick. As the organism grows on the agar, you can transfer a small piece of it to a new agar plate to start a new culture. Repeat this process until you have a sufficient number of cultures to transfer to a larger container for further cultivation. By following proper sterilization and transfer techniques, you can successfully use agar to cultivate mushrooms indefinitely.
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