Agar work is the foundation upon which everything is built for many cultivators. Petri dishes allow you to isolate pure cultures, store mushroom strains, and monitor the condition of your cultures precisely. The visual signs of mycelium growth help you to detect contaminations early and react accordingly.
The classic recipes
The classic recipes have proven themselves. Malt extract agar (MEA) is universally applicable and promotes excellent mycelium growth. As a rule of thumb for quantities, calculate about 20 grams of malt extract (around 2%), 15–20 grams of agar (around 1.5–2%), and 3–4 grams of yeast extract per liter; fill up the rest with distilled water. For four liters of MEA, you therefore need around 80 grams of malt extract and 60–80 grams of agar. Potato dextrose agar (PDA) is also popular, especially with moisture-loving mushrooms.
Important note on agar quantity: Agar must be used in a sufficient concentration (about 1.5–2% or 15–20 g per liter) for the medium to gel firmly. Significantly lower amounts – for example, only 15–20 g for four liters – do not result in a stable gel and are unsuitable for plate cultures.
Everything is stirred in a pot and heated slowly until the agar dissolves completely (do not boil!). Subsequently, the medium is sterilized. In this context, "sterilizing" does not mean simple heating, but a steam pressure sterilization in an autoclave or pressure cooker – standard conditions are approx. 121 °C at around 15 PSI for about 15–30 minutes. Only this effectively kills heat-resistant spores; mere boiling is insufficient and almost always leads to contaminated plates.
⚠️ Safety instruction – Steam pressure (autoclave/pressure cooker): Hot, pressurized steam is generated during sterilization. There is a risk of scalding and bursting.
- Only use intact, pressure-tested equipment and follow the pressure cooker/autoclave manufacturer's instructions.
- Check the pressure relief valve and seal before every use; never force the device open.
- Let the pot cool down completely and release the pressure entirely after sterilization before opening it. Do not direct hot steam toward your face; wear heat-resistant gloves.
Pouring plates
Pouring plates requires some skill, but is quickly learned. After sterilization, let the medium cool down just enough so that it is still liquid but no longer boiling hot – ideally brought to about 50 °C in a water bath. Then fill the Petri dishes with agar to about 20–25%: slightly tilt the dish with the palm of your hand, pour in the liquid agar, and immediately return it to a horizontal position. The agar solidifies at room temperature within a few minutes. Stored in a dry place protected from light, the finished plates can be kept for weeks to months.
Transfers and isolations
Transfers and isolations are the core skills. To obtain a pure culture, take a small piece of mycelium from your source (using a sterile inoculation loop or scalpel) and place it on the new plate. Under ideal conditions – 20–25 °C, darkness or dim light – the mycelium will grow into a beautiful white colony within 7–14 days.
If there are multiple mycelium fronts on a plate, you can work with serial dilutions: cut a tiny piece from the furthest, clean growth front, transfer it to a fresh plate, let it grow again, and repeat the step. With every transfer, you move further away from potential contaminants. After three to four generations, you will generally obtain a practically pure culture.
Clean or contaminated? How to tell the difference
Distinguishing between a clean and a contaminated culture is usually done visually and by smell. Pure mycelium is white or creamy, spreads evenly, and smells neutral or pleasantly mushroom-like.
Contaminations, on the other hand, often appear colored or exhibit conspicuous structures:
- Trichoderma (green mold): starts white and quickly turns a vibrant green (light green to dark green) as it sporulates; spreads aggressively and rapidly.
- Cobweb mold (Cladobotryum/Dactylium): forms loose, gray-silvery, spiderweb-like threads that cover the surface very quickly.
- Bacterial contamination (e.g., Bacillus, "wet spot"): appears as slimy, wet, shiny spots with an often acidic odor – not as a color.
- Neurospora (orange/pink bread mold): stands out due to a characteristic orange to pink discoloration and spreads extremely quickly.
Caution: A white, fluffy coating is not automatically healthy mycelium – some molds also start out white. The decisive factors are the subsequent sporulation (color), the growth pattern, and the smell. Unknown impurities are typically colored, spread rapidly, and smell musty or unpleasant. In case of doubt: discard the plate and approach the next attempt even cleaner and more sterile.