The vegetative body of a fungus, known as mycelium, is a network of fine, thread-like structures called hyphae. Growing this mycelium on a nutrient medium is a foundational technique in mycology, allowing for the precise management of fungal genetics. Agar, a gelatinous substance derived from red algae, provides a transparent, solid surface that is mixed with nutrients like malt extract or potato dextrose to support fungal growth. This method allows cultivators to isolate pure cultures, purify strains from contaminants, and clone the desirable characteristics of a specific mushroom specimen. Working with agar provides a controlled environment to observe and select the most vigorous and contamination-free fungal strains before scaling up cultivation.
Essential Preparation and Sterilization Setup
A clean workspace is paramount because the nutrient-rich agar medium is attractive to airborne spores and bacteria. The most common solution for creating a sterile environment at home is the Still Air Box (SAB), a simple plastic container with armholes that minimizes air currents and allows contaminants to settle. For more advanced work, a Laminar Flow Hood (LFFH) provides a continuous stream of filtered, sterile air, offering a higher level of protection.
All surfaces within the work area must be thoroughly wiped down with a 70% isopropyl alcohol solution. This is part of aseptic technique, which maintains a sterile environment to prevent contamination. Tools, such as scalpels or inoculation loops, must also be sterilized between every use.
Flame sterilization is the common method for metal tools, involving heating the blade or wire until it glows red hot. This intense heat incinerates any microbes or spores. The tool must then be allowed to cool completely before touching the living mycelium or the agar, as heat will kill the fungal tissue. A common cooling method involves briefly pressing the hot scalpel tip into a clean section of the agar medium, away from the inoculation site.
Techniques for Inoculating Agar Plates
Inoculating an agar plate is the process of transferring fungal material onto the sterile nutrient surface, requiring precision and speed within the sterile workspace. One common approach is inoculating from a spore syringe, where one or two drops of the spore-suspended liquid are gently placed onto the center of the agar. Spores are genetically diverse and may introduce contaminants, making the agar plate an excellent testing ground for germination and purification.
Cloning a mushroom, known as tissue culture, involves transferring a small piece of the fruiting body’s inner flesh to the agar. The mushroom is torn open rather than cut to minimize surface contamination. A tiny piece of inner tissue, about the size of a grain of rice, is carefully excised with a sterile scalpel and placed onto the center of the plate.
Transfers are also made from one culture to another, known as an agar-to-agar transfer, to isolate the best genetics or clean up a culture. A sterile scalpel is used to cut a small, typically triangular wedge from the leading edge of healthy mycelial growth. This wedge is quickly transferred face-down to the center of a fresh agar plate. Transfers from sources like colonized grain spawn or liquid culture follow a similar principle of minimal exposure and rapid placement onto the new medium.
Incubation and Contamination Management
After inoculation, the sealed plates are placed in an incubation environment, ideally a dark area with a stable temperature range of 70 to 75°F (21 to 24°C). Within a few days to a week, the mycelium will begin to colonize the plate, radiating outward from the inoculation point. Healthy growth can appear in two primary forms: rhizomorphic, characterized by thick, rope-like strands, or tomentose, which presents as a fluffy, cotton-like mass. The transparent nature of the agar is useful for spotting contamination.
Contamination Identification
Green mold, often Trichoderma, is a rapid competitor that initially appears white but quickly turns a dusty green as it sporulates. Bacterial contamination typically manifests as a wet, shiny, or slimy film, sometimes appearing yellowish or gray. The presence of bacteria is often betrayed by a foul, sour, or musty smell when the plate is momentarily opened. Yeast contamination appears as distinct powdery or bubbly colonies, often concentrated near the inoculation point.
A contaminated plate should be immediately sealed and isolated from clean cultures to prevent the spread of spores. If the desired mycelium is growing vigorously and the contamination is localized, a small piece of clean mycelium can be subcultured from the farthest leading edge to a new plate. This cleaning process often requires multiple transfers to achieve a completely pure culture.
Using and Storing Mycelial Cultures
Once an agar plate is fully colonized with a pure, healthy culture, it is ready to be expanded for a larger-scale grow. The clean mycelium is typically transferred directly to sterilized grain spawn, which serves as the primary inoculum for bulk substrate. Alternatively, a small agar wedge can be used to inoculate a sterile liquid culture, creating a large, injectable volume of active mycelium suspended in a nutrient broth.
For long-term preservation, the colonized agar plates can be sealed with Parafilm, a stretchy material that seals the plate while allowing for necessary gas exchange. The sealed plates are then stored in a refrigerator at approximately 4°C. This cooler temperature drastically slows the mycelial metabolism, allowing the culture to remain viable for many months.