Sarah Brown
Agar is a gelatinous substance derived from red seaweed that is commonly used in mushroom cultivation. It is a versatile and vital growth medium that provides a stable, controlled, and observable environment for the growth and study of fungi. Agar's transparency and resistance to contamination make it ideal for cultivating mushrooms. In this process, agar is infused with nutrients, poured into petri dishes or jars, and inoculated with mushroom spores or mycelium samples. Proper incubation and maintenance of a sterile environment are critical for successful mushroom cultivation with agar.
| Characteristics | Values |
|---|---|
| What is agar? | A gelatinous substance derived from red seaweed |
| Why use agar? | To clean up contamination, isolate genetics, and cloning |
| How to use agar? | Cut some colonized agar and put it on a new agar plate. |
| How to make agar? | Add a food source to agar to create a nutrient-rich agar media for mushroom cultures. |
| How to sterilize agar? | Place the jar into a pressure cooker or a large pot with water. Sterilize for 45 minutes. |
| How to incubate agar? | Place inoculated agar plates in a clean, sealable container at a temperature ranging from 75-80°F (24-27°C). |
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What You'll Learn
Agar preparation
Agar is a gelatinous substance derived from red seaweed that is used in mushroom cultivation. It is primarily used in petri dishes or jars to germinate spores and culture mycelium. The clear, nutrient-rich medium allows cultivators to observe the growth and health of the mycelium, isolate pure cultures, and identify any contamination.
There are various types of agar media, each formulated to serve different purposes in mushroom cultivation. One of the most common types is Light Malt Extract Agar (LMEA), made from light malt extract, agar-agar, and water. It is suitable for a wide range of mushroom species. Other types of agar mix include PDA (potato dexrose agar), MYA (malt yeast agar), and DFA (dog food agar).
To prepare agar for mushroom cultivation, follow these steps:
- Prepare the agar mix by combining the desired ingredients, such as malt extract, yeast extract, or other nutrient sources, with agar-agar and water. Ensure that the malt extract is diluted, as it can get stuck to the jar.
- Place the mixture into a jar and screw the lid on tightly.
- Sterilize the jar using a pressure cooker or a large pot with water. If using a pressure cooker, place the jar into the cooker or pot with water reaching the 2/3 level of the jar. Lock the lid and sterilize for 45 minutes at 121°C/ 250°F/ 15 psi/ 1.05 bar. For those without a pressure cooker, boiling the jar on the stove for around 2 hours is an alternative method.
- After sterilization, remove the jar from the heat and allow it to cool. The ideal pouring temperature for the agar mixture is around 46-52°C (115-125°F), ensuring it remains liquid for easy pouring without compromising sterility.
- Once the mixture has cooled, pour it into petri dishes or jars, where it will solidify to form a growth medium.
- Introduce spores or mycelium fragments onto the agar surface. You can do this by slicing pieces of a mushroom stem and placing them onto the agar mixture, ensuring the lid is on to prevent dust from entering.
- Store the dishes in a warm and dark place, ideally around 24°C (75-80°F).
It is important to maintain a clean and sterile environment throughout the process to prevent contamination. Regularly monitor the plates for signs of mycelial growth and potential contamination. With proper incubation and care, you can successfully cultivate mushrooms using agar preparation techniques.
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Incubation
During incubation, it is important to regularly monitor the plates for signs of mycelial growth and potential contamination. This can be done visually due to agar's transparency. If any contaminants are spotted, it is advisable to remove the affected plate to prevent the spread to other plates. The mycelium will usually take several days to a couple of weeks to fully colonize the agar, depending on the mushroom species and the specific incubation conditions. Once the agar is fully colonized, you can transfer the mycelium to a substrate or directly to a fruiting chamber.
Agar media come in various types, each formulated to serve different purposes in mushroom cultivation. Understanding the different types of agar media and their uses can increase your success rate. For example, Light Malt Extract Agar (LMEA) is a common homemade alternative that is suitable for a wide range of mushroom species. Other types of agar media include Potato Dexrose Agar (PDA), Malt Yeast Agar (MYA), and Dog Food Agar (DFA).
Proper incubation is key to successful mushroom cultivation. By mastering this stage, you will set the foundation for robust and healthy mycelial growth.
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Contamination
The human body is the greatest source of contamination in mushroom cultivation. Human hands and clothes contain lots of bacteria and spores that can ruin a crop. Before you get to work, take a shower and use hand sanitiser. Clean clothes, including a lab coat or scrubs, can be helpful in addition to a face mask or mouth covering and laboratory gloves. Not talking when working will help you limit contamination, especially when near open containers such as plates, jars, or substrate blocks.
The air is also a very likely source of contamination for mushroom growers. Mould spores and other contaminants, although invisible to the naked eye, are everywhere, just waiting to ruin your project! The best option to reduce contaminants in the air is to use a laminar flow hood, which is a workbench that includes a filter pad, fan, and a HEPA (High Efficiency Particulates Air) filter built into it. The system is costly but worth the investment for medium to large-scale culture plate work and liquid culture inoculations. If a flow hood is not in your budget, the next best option is a still air box, which works well for small-scale projects and is a popular choice for home cultivators.
A common reason for contamination in mushroom cultivation is incomplete sterilisation or improper pasteurisation of the bulk substrate before inoculating with a mushroom culture. The bulk substrates and grains naturally contain competing organisms and dormant spores that need to be reduced or eliminated, otherwise, they will try to out-compete your mushrooms. Supplemented sawdust fruiting blocks and mushroom grain spawn need to be completely sterilised before inoculation. This means subjecting the substrate or grain to high pressure and temperature for an extended period of time. The best way to do this is to use a stovetop pressure canner that is big enough to hold the material and can reach 15 PSI.
Agar, a gelatinous substance derived from red seaweed, plays a crucial role in mushroom cultivation. It is primarily used in petri dishes or jars to germinate spores and culture mycelium. The clear, nutrient-rich medium allows cultivators to observe the growth and health of the mycelium, isolate pure cultures, and identify any contamination. Black agar, for example, aids in spotting contamination early, ensuring cleaner cultures. While the above agar media are commercially available, homemade alternatives are also possible for mushroom cultivation. The most common is Light Malt Extract Agar (LMEA), made from light malt extract, agar-agar, and water. It is suitable for a wide range of mushroom species.
To make sterilised agar plates, you can use a variety of recipes, including PDA (potato dexrose agar), MYA (malt yeast agar), and even DFA (dog food agar). They all consist of adding some sort of food source to agar to create a nutrient-rich agar medium for mushroom cultures. The mix that is most used is Malt Extract Agar. Once you have nutrient-rich agar media, you can use it to propagate cultures, store cultures for long-term use, germinate spores, and even clone mushroom fruit bodies.
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Isolation
Selecting the Mycelium:
Begin by examining the agar plate and identifying the most robust and healthy-looking mycelium. Look for strong and fast-growing mycelium with prominent ropey rhizomorphic growth rather than fluffy tomato-like growth. These characteristics indicate active and vigorous mycelium that is ideal for isolation.
Isolating the Mycelium:
Once you have identified the desired mycelium, carefully cut or slice a piece from the selected growth. Choose a section near the edge of the healthiest growth, as this area is likely to be the most active and contaminant-free. Transfer this piece of mycelium to a new, sterile agar plate. This process creates a clone of the original mycelium and ensures that you are working with a pure culture.
Incubating the Isolated Culture:
After transferring the mycelium to a new agar plate, place the plate in a clean, sealable container such as a plastic tote or a specialized incubator. Maintain a consistent temperature range of 75-80°F (24-27°C) for most mushroom species. You can use a heating mat, temperature controller, or a warm room to achieve this temperature. Proper incubation provides an optimal environment for the isolated mycelium to thrive and colonize the new agar plate.
Monitoring and Contamination Control:
Regularly monitor the agar plates during incubation. Check for signs of mycelial growth and potential contamination. If contamination occurs, remove the affected plate immediately to prevent the spread to other plates. Isolation improves the chances of obtaining pure cultures, but contamination can still occur. By monitoring closely, you can catch any issues early and take appropriate corrective actions.
Long-Term Storage:
Once the mycelium has fully colonized the agar plate, you can transfer a sample to a culture slant for long-term storage. This step ensures that you can preserve the isolated culture for future use and continue working with a pure strain of mycelium.
By following these isolation steps, you can effectively select, separate, and maintain pure cultures of mycelium, which are essential for successful agar mushroom cultivation.
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Storing cultures
Storing mushroom cultures for long-term use can be done by inoculating agar plates with a liquid syringe. The syringe tip should be flame-sterilised until red-hot before transferring a small amount (1-1.5 CC) of the culture to the centre of the dish. The plates should be kept level to prevent the liquid culture from flowing out. The culture will be ready for long-term storage or transfer in about 7-10 days, depending on the species.
Culture slants are another way to store mushroom cultures long-term. Slants are test tubes filled with mushroom agar that gives the mycelium more surface area to grow on. To prepare a slant, fill a test tube with your chosen agar mix, sterilise it in a pressure cooker, and let it cool down at an angle so the agar slants. Then, add some healthy mycelium to the agar and seal the test tube before storing it in the fridge (between 4-10°C).
Most cultures can be stored in the refrigerator, where they will remain viable for a long time, up to a few years or more. However, some cultures, such as pink oyster mushrooms, will likely die off if placed in the fridge. Therefore, it is important to check which method of storage is appropriate for your particular culture.
Another method for storing mushroom cultures is to use sterile distilled water. This method involves storing the culture at room temperature away from direct sunlight. The mushroom culture enters a dormant state and is held in stasis. However, if conditions deteriorate and the room becomes too hot, the vials can be refrigerated.
Additionally, liquid nitrogen can be used to store cultures at -384°F (-196°C), essentially stopping all metabolic activity and allowing for indefinite storage. Alternatively, a chest freezer can be used to store cultures at -5°F (-18°C). The culture is mixed with glycerol to prevent ice crystals from damaging it, and it is stored as a frozen mush in slants.
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Frequently asked questions
Agar is a gelatinous substance derived from seaweed, specifically a group known as red algae. It is an ideal growth medium for fungal mycelium, offering a sterile and nutrient-rich environment for development.
Agar is used to clean up contamination, isolate genetics, and cloning. It is also used to germinate spores and culture mycelium.
First, sterilise your tools, substrates, and agar. Then, prepare your agar mixture, ensuring the malt extract is diluted. Place the mixture into a pressure cooker or large pot with water and sterilise for 45 minutes. Once cooled to around 46-52°C, pour the mixture into your chosen container.
Once the agar is fully colonised, transfer the mycelium to a substrate or directly to a fruiting chamber.
