Agar petri dishes (plates)
Heating liquid culture
“Mushrooms reproduce through spores. In the highly competitive natural world, the chances of mushroom spores germinating and then producing a mushroom are slim. Within a laboratory, isolated from airborne contamination, the probability of success is much improved. What a cultivator does is remove a select species from the fierce competition of outdoors into an optimized environment indoors wherein the mushroom mycelium grows unhindered from the ravages of nature. This harbor of quiet refuge is, in effect, the sterile laboratory.”
–Paul Stamets, A Simplified Overview of Mushroom Cultivation Strategies, 1996
Using simple techniques at home, you too can cultivate and keep edible gourmet and medicinal mushrooms for access any time. In this Lab Skills lesson, Jonathan Carver, our own lead mycologist, demonstrates how to produce growth media for mushroom cultivation using plates, slants and liquid culture. We covered the techniques you would use for each method along with their applications and why you would choose one over the other. We worked in the sterile confines of the lab, but many of these same techniques can be adapted to a home setting.
Note: If you don’t want to produce your own media, you can buy premade agar plates and slants by clicking on the links below.
Intro to Plates, Slants, and Liquid Culture
Plates are the work surface for working with fungi. They allow you to observe growth patterns, speed of colonization, and give you the opportunity to separate different mycelium strains taken from the spores of a single mushroom. Plates are useful for cloning wild mushroom strains, isolating specimens away from contaminants, and conducting mating trials between different strains. Plates contain solid media culture consisting of natural sugars and agar, a jelly-like substance derived from red algae that gives the mycelium an anchor while we do our work.
Slants, on the other hand, are ideal for long-term culture storage. The narrow opening of a test tube is a little harder to work with but less apt to capture contamination and allows you to culture a fungus on a small amount of solid media, place it in the fridge, and store it more or less indefinitely until you need it. Often, you’ll store your culture on slants and transfer a small portion of culture from the slant to a culture plate to begin work on it.
Liquid culture is often how the public first encounters mushroom cultivation. Liquid culture is less susceptible to airborne contaminants, as it is housed in a sealed container such as a syringe, and ideal for working with fungi at home or a slightly less sterile environment. Because the mycelium is in a nutrient broth, it can contain lots of small hyphal fragments which can each serve as inoculation points when you use the liquid culture to expand onto other substrates. Liquid culture tends to colonize whatever substrate you’re trying to grow on a little bit faster than when you work with a plate.
Making Media for Plates, Slants, and Liquid Culture
Initially, the preparation for making culture media is similar for plates, slants and liquid culture, but as we progress toward later steps, some of the techniques diverge. For plates we’ll use the culture media MYA, short for “malt, yeast, agar” along with a jar of PDA, short for “potato, dextrose, agar” for making slants. In MYA, malt extract provides sugars and carbohydrates while yeast provides additional nutrients. In the PDA solution, dextrose serves as the primary food source for the growing mycelium while the potato extract provides extra nutrients. In both preparations, agar solidifies the media. For liquid culture, we’ll use a simple combination of malt and yeast. Once each solution is prepared we’ll pasteurize them in a pressure cooker to ensure all rival microbes are eliminated.
- Graduated cylinder
- A flask, Pyrex bottle, or a mason jar with an injection port lid (for liquid culture)
- Hot plate with stir bar
- A pressure cooker (such as an All American)
- 500ml of water
- 10 grams of malt
- 10 grams of agar
- 1 gram of yeast
- 750ml of water
- 10 grams of dextrose
- 10 grams of agar
- 2 grams of potato extract
- Alternatively 30 grams of PDA pre-mix
- 500ml of water
- 10 grams of malt
- 1 gram of yeast
Step 1) Fill your flask, Pyrex bottle, or mason jar with the appropriate amount of water and place on a hotplate. Seal your container but do not over tighten. This allows any pressure or condensation to escape. Heat to a boil.
Step 2) While the water is heating up, measure out your dry ingredients and add them to the individual appropriate flask, Pyrex bottle, or jar following the recipes above. Wait for the mixture to fully combine in solution and heat to temp. An optional step is to use a biological indicator to confirm your media is properly sterilized.
Step 3) Once your solution is ready, prepare the pressure cooker. We used an All American pressure cooker, but you can use any pressure cooker you may have at home for this step but ensure you follow the instructions for the particular model. After, take your solution, double check that it’s loosely lidded, and place it inside. Include any autoclavable bags you’ll be using to store the plates once they've been poured. Close the pressure cooker properly by making sure each screw is balanced.
Step 4) Set a timer for 18 minutes (at this point you’ll see steam rising out). Once the timer goes off, open the pressure cooker for a few minutes to air out. After, seal it again and let it go for another 18 minutes. Pressure cookers vary, so make sure you follow the instructions to ensure you’re properly cooking your media.
Flow Hood Note
A clean environment is essential for working with culture media, because it is very prone to contamination. That being said you can do some of these same techniques at home by building your own flow hood, glove box or still air box (SAB) which are simple structures that allow you to create a microenvironment that is very clear. It is not the recommended technique but you may even pour plates and conduct culture work in the open air but your success rate will be very small.
Step 5) This process requires a flow hood. Sterilize your space, including your flow hood, gloves, and plates. Remove your MYA media from the pressure cooker (make sure the pressure is down), and place it inside the flow hood. Be careful as your media may be very, very hot. Allow it to cool until it’s comfortable to touch.
Step 6) Once your media is ready, it’s time to pour. If it’s still too uncomfortable to touch, you can wrap a sterile paper towel around the bottle. As you begin to pour, start from the bottom of the stack of petri dishes and make your way up to the top in order to minimize the time that the inside of the plate is exposed to open air. Continue this process until you’ve used up all of your media.
Step 7) Allow your plates to cool inside the flow hood. Once they're solid, seal them inside of a sterile bag, and store them at room temperature. You can also store them inside of the fridge as well. Shelf life is approximately 2 months.
Pouring agar into petri dishes
Culture grown in a slant
Step 1) To make slants, use a pipette to transfer 20 millimeters of PDA media into each glass tube. Make sure to loosely cover each glass tube.
Step 2) Pressure cook your tubes to 15 PSI. Set between 15 to 20 minutes. Again, we used an All American pressure cooker, but you can use any pressure cooker you may have at home for this step but ensure you follow the instructions for the particular model. Add to the pressure cooker any autoclavable bags you may use to store your test tube rack once fully pasteurized.
Step 3) Take your tubes out of the pressure cooker and refrigerate for at least 4 hours, allowing the media to solidify at an angle or “slant” by leaning the whole rack onto an object such as a small scale to ensure the entire batch solidifies the same way. This creates a larger surface area for the fungal culture at the surface of the media. Allow caps to stay loose while the media cools to allow for condensation to evaporate but tighten the seals once it has solidified to store until you’re ready to use them.
Liquid Media Prep
Once the liquid media is fully pasteurized in the pressure cooker it is ready for inoculation once it has cooled down. Keep in mind proper sterile techniques when using culture syringes for inoculation of liquid culture media.
Amendments, Antibiotics and Strain Senescence
It’s recommended to swap out media every so often to avoid strain senescence. MYA and PDA can be used interchangeably between plates and slants, whereas liquid culture may benefit from the addition of amendments depending on the application.
Strain senescence is the gradual decline in vigor of a particular mushroom culture over time. By alternating media, you’re introducing new carbohydrates which help activate the production of different enzymes and maintain strain viability.
Some mycologists may also add amendments to media such as: peptone, mineral gypsum, or small fragments of substrate, which can help improve mycelial growth, avoid strain senescence and, in some cases, train certain types of mushroom cultures to more effectively break down a specific food source.
Certain antibiotics, such as ampicillin and streptomycin, can also be added to media to suppress bacterial growth when isolating strains from the wild or cleaning up a contaminated culture. Keep in mind that antibiotics should only be used when you’re working with a particularly contaminant prone culture. In addition, depending on the antibiotic used, you may have to add them before or after cooking your media.
Tips for Using Plates, Slants, and Liquid Culture
By transferring a culture from slants to plates, you’ll be able to better work with your culture. Whether you're studying the morphology of a culture, propagating it onto spawn, or just trying to clean up a culture that has some contamination, plates are essential when doing lab work.
Tips for transferring from slants to plates:
- You only need a small tissue sample, the size of a grain of rice, to inoculate a plate.
- Be sure to place your tissue sample in the center of the agar plate and seal it immediately by using parafilm. This helps reduce contamination.
- If you’re taking multiple samples, make sure to sterilize your tools before and after each use.
- Once you have a stack of inoculated plates, you can store them inside of an autoclavable bag.
- Once your plates are inoculated, leave them at room temperature to allow the mycelium to grow out on the surface of the plate.
- Once your culture has completely grown out to the edge of the plate, you can store them for 1-3 months in a refrigerator.
Transfering from Plates to Slants or Liquid Media
Once you have a colonized plate, you can either transfer it onto slants for long-term storage or use it to inoculate liquid culture media.
Sampling culture from a slant
Culture placed in an agar petri dish
Tips for transferring from plates to slants or liquid media:
- When taking sections of mycelium you’ll want to take from the center area of the plate. You don't want to be near the middle of the plate, nor at the very edge of the mycelium as these areas are more likely to have contaminants.
- As with plates you should seal agar slants with parafilm, and leave them to incubate at room temperature. Once the mycelium has completely grown over the surface of the slant, you can store them in a fridge for up to a year.
- You can also use plates to inoculate liquid media. Once inoculated, mycelium will take a couple of weeks to grow throughout the media. When fully colonized, liquid culture can be used to create spawn or more liquid culture.
Inoculating Liquid Media with a Culture Syringe
Inoculating liquid media with a culture or spore syringe is the most common technique used by home scale or low-tech cultivators. Some commercial growers use this technique as well, as it limits the possibility of contamination, and may be easier to work with.
Tips for inoculating liquid media with a culture syringe:
- Although this technique may work well in non-sterile environments, it’s recommended to conduct inoculation in either a still air box or flow hood to reduce the possibility of contamination.
- Be sure to sterilize your needle before use.
- It may be more difficult to spot contamination in liquid culture.
Working with plates, slants and liquid culture at home can be extremely rewarding. Developing these unique skills will allow you to cultivate and enjoy edible and gourmet mushrooms year-round, increasing your self-sufficiency while also saving you money. You may even learn something new about the natural world while doing so!
A Simplified Overview of Mushroom Cultivation Strategies