Mycelium is the vegetative body of a fungus, forming a vast network of thread-like filaments called hyphae, which function much like a root system. As the foundation for all fungal growth, preserving healthy mycelium cultures is necessary for mycologists, researchers, and commercial cultivators. Maintaining these living strains in culture banks prevents the natural process of senescence, or genetic degeneration, that occurs with repeated transfers and aging. Simply placing a culture in a standard freezer is lethal to the cells, necessitating the specialized technique known as cryopreservation.
Why Simple Freezing Fails
The primary challenge in freezing biological tissue is the high water content within the cells. Uncontrolled freezing allows the water to crystallize, which physically destroys the delicate cellular structures. These sharp, jagged ice crystals puncture cell walls and internal organelles, causing irreversible mechanical damage.
A second damaging event is the osmotic shock that occurs as water freezes outside the cells. As pure water turns to ice, the remaining unfrozen solution becomes highly concentrated with salts and solutes. This extreme concentration gradient pulls water rapidly out of the mycelial cells in an attempt to equalize the pressure, leading to severe dehydration and cellular collapse.
Cryopreservation Techniques for Mycelium
Successful cryopreservation requires a multi-step process designed to shield the mycelial cells from freezing injury. The culture is first prepared, often by growing the mycelium in a liquid broth or by cutting small, active agar plugs from the growing edge of a colony. These small samples are then transferred into specialized cryovials.
The introduction of a cryoprotectant agent is the most important step for cellular protection. Chemicals like glycerol, typically used at a concentration of 10% to 20%, or dimethyl sulfoxide (DMSO) are added to the cryovials. These agents penetrate the cell walls, lowering the freezing point of the water and preventing the formation of large, destructive ice crystals. Certain sugars, such as sucrose or glucose, can also be effective cryoprotectants, particularly when the mycelium is grown on a grain substrate.
Following the addition of the cryoprotectant, the vials must be cooled at a slow, controlled rate. A common method involves placing the vials into an insulated container, such as a specialized slow-freezing device, and then moving this container to a -80°C freezer. This insulation achieves a controlled cooling rate of approximately 1°C per minute, minimizing cellular stress. For the most stable, long-term storage, the vials are transferred to the vapor phase of liquid nitrogen, which maintains a temperature of -130°C to -196°C.
Reviving and Assessing Frozen Cultures
When a strain needs to be retrieved from storage, the thawing process must be executed rapidly to prevent a phenomenon known as recrystallization. If thawing occurs slowly, the small, harmless ice crystals formed during controlled cooling can grow into larger, damaging crystals as the temperature rises toward the melting point. To counteract this, the cryovials are removed from ultra-low storage and immediately placed into a warm water bath, often maintained between 30°C and 37°C.
Once the sample is completely thawed, the mycelial suspension or agar plugs are quickly transferred to a fresh, nutrient-rich growth medium. The revived culture is then incubated under optimal conditions. An initial observation period is necessary because the mycelium may exhibit a prolonged lag phase, meaning it takes longer to resume growth than a non-frozen culture.
The final step involves assessing the culture’s viability and purity. Viability is confirmed by observing the vigor and speed of colonization on the new medium. Functional tests are performed to confirm genetic stability, ensuring the revived culture has maintained its original characteristics, such as normal morphology or expected enzyme activity.