by Michael Schatte
Watch our YouTube video on how to inoculate Agar and Grain!
Need agar plates?
Agar Plates are petri dishes that contain water, agar and nutrients. This combination provides a gel like substance that’s highly nutritious for fungi. It’s also very beneficial for molds, yeasts and other microorganisms. The Agar Plates you have received have been sterilized in a commercial steam autoclave to ensure no living organisms or spores are in the mixture.
Important Info! Agar Plates are highly susceptible to contamination from molds spores, bacteria and yeasts floating in the air around us. When growing from spore syringe/spore prints/liquid culture you must ensure there are no contaminants within them. This can sometimes be impossible to know. Agar to agar transfers can be very beneficial in isolating away from contamination. This may take several transfers to obtain a completely clean culture.
Contamination Vectors
Contamination is any microorganism (mold, bacteria, yeasts, fungi) that’s not your intended fungi. When working with agar or any highly nutritious medium it’s important to consider all possible sources of contamination. Most of these sources of contamination can be minimized to acceptable levels.
- Inoculum - Spore Syringe/Prints/Liquid Culture – Ensure you use a trusted vendor with a reputation of providing clean inoculum. However, contamination can still happen.
- Clone Specimen – If you are cloning a cultivated/wild mushroom it’s important to understand that these will more than likely contain contaminants. Cloning mushrooms on agar should be considered a process since it may involve multiple transfers (agar to agar) to end with a clean culture.
- Tools – All tools (scalpels, inoculation loops, etc) must be sterilized via a pressure cooker or flame sterilization. Using a butane torch you can heat these metal tools until red hot to ensure sterilization between inoculation/transfers.
- Air – The air around you can contain 1,000 to 10,000 fungal spores per square meter. It’s critical that you account for this by using either a flow hood or Still Air Box (SAB)
- You – Yeasts, bacteria, mold and fungus spores will land on your skin and hair throughout the day. You may unknowingly contaminate your agar plates by simply waving your unwashed hand or arm over the plates. It’s highly encouraged to work with nylon gloves since they can be sanitized via alcohol before work with the plates. Personal hygiene can have a big impact on your success.
- Hole/gap in Parafilm Tape – After inoculating your agar plates, you must use the enclosed Parafilm to seal the plate to protect against contaminants. This tape MUST cover the entire edge of the plate with no gaps or holes.
A Still Air Box (SAB) can be constructed out of a clear 56 qt plastic tote. Simply cut two holes just big enough to place your hands inside. Use isopropyl alcohol (Lysol may also work) to clean in the inside of the tote. The idea is to limit the amount of air exchange. This will help to ensure there are no contaminants being pulled into your work area.
NEVER use an open flame around alcohol!
Inoculating the Agar Plates
Need agar plates?
It’s highly encouraged to use with a flow hood or Still Air Box (SAB) when working with open agar plates. These will help to ensure success in minimizing the air contamination vector.
- Remove the agar plates from the sealed bag within your flow hood or SAB.
- Stack them up and work with one open plate at a time.
- Spore Syringes – Place a tiny drop (<1cc) on the center of the plate. Use a sterile scalpel, inoculation loop or simply move the plate around to spread the liquid around.
- Spore Print – using a sterile scalpel/inoculation loop scrape a small amount of spores onto the agar surface.
- Liquid Culture – use the same Spore Syringe method on previous page.
Using Parafilm can be a little tricky to get right without ripping it. Pick up the plate and hold with your left hand (if right-handed) with the edge of the plate facing you. Hold the parafilm strip with your left thumb on the outer edge of the plate. Using your right hand stretch the film around the agar plate in 1-2 inch sections. Work around the plate in this fashion. If the parafilm breaks, simply place the broken portion on top of the parafilm left on the plate and continue around the agar plate.
- Cloning – 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 that agar surface.
- Once completed, place the lid on the agar plate and use the included Parafilm to seal the plate.
Incubation
Agar plates can be incubated at room temperature for most species. Incubation chambers can be used to increase the temp to encourage germination. However, be mindful of the grow parameters of the specific species you are working with.
Starting with spores can take up to 6-8 weeks to see any growth on agar. Spore germination, hyphae growth and fusion into mycelium can take some time and luck to get right. Once noticeable growth begins you will start seeing signs of sectoring. This typically indicates varying genetics due to different spores containing varying genetic characteristics. You may choose to isolate certain sections based on vigorous growth of the mycelium.
Visible Signs of Contamination
Mycelium (the white vegetative growth of fungi) will begin to spread out across the agar plate. It will have the appearance of a white fuzziness originating from the area of inoculation. Molds, bacteria, and yeasts will appear on the plate in small spots but will spread quickly.
- Yeasts/bacteria will typically have a white/yellowish milky appearance.
- Molds will have a somewhat similar look to mycelium but will spread very quickly and will turn green or black.
- Avoid opening agar plates that have dark green, white or black powdery appearance around clean agar plates.
Agar to Agar Transfers
Once the mycelium has grown to about 80-90% of the agar plate, you can choose to transfer the culture to more agar plates. You may also transfer uncontaminated sections of contaminated agar plates. Transferring away from contamination allows you to obtain a clean culture. Multiple transfers may be necessary to achieve a completely clean culture. Transferring from agar to agar is done using the following procedure.
- Choose the best possible candidate to transfer from. This may be the least contaminated or the most vigorous growth section.
- Working inside your flow hood or SAB open the colonized plate.
- Remove the parafilm tape from the clean agar plate, leaving the lid in place.
- Flame sterilize your scalpel until red hot.
- Cut a small 1x1 cm square from the leading each of the mycelial growth.
- Using the scalpel pick up the mycelium square, open the clean agar plate and place the mycelium square in the middle of the clean plate, close the lid.
- Use Parafilm, plastic wrap, Glad Press’n Seal and even blue painters tape can be used to seal the agar plates.
Agar plates can be stores in a sealed container within your refrigerator to slow growth once a clean culture is obtained.
