Mycelium is the vegetative body of a fungus, a dense network of thread-like cells called hyphae that function as the growth engine. To cultivate and study this network outside of its natural environment, mycologists use agar, a gelatinous substance derived from seaweed that is mixed with nutrients like potato dextrose or malt extract. This nutrient-rich gel provides a solid surface for the mycelium to colonize and grow rapidly in a laboratory setting. While this technique allows for clean isolation and propagation, the agar medium is only a temporary home for the fungus, making the knowledge of its lifespan a necessity for strain maintenance.
The Standard Time Window for Agar Cultures
Mycelium stored at ambient room temperature (20°C to 25°C) usually remains vigorous for one to two months before showing signs of decline. This higher temperature encourages faster growth, which accelerates the consumption of the limited food source. A colonized agar plate is considered a short-term holding method, with viability measured in weeks to a few months.
To slow down fungal metabolism, plates are typically stored under refrigeration (4°C to 8°C). Lowering the temperature decreases the rate at which the mycelium consumes nutrients, extending its usable life to three to six months. The exact duration depends on the specific fungal species or strain, as some robust varieties last longer than others.
Once the mycelium completely colonizes the plate, it enters a state of slowed growth, leading to eventual decline. Periodic transfer to a fresh plate is the only way to maintain a culture indefinitely without using more specialized preservation methods.
Factors Limiting Mycelial Viability
The primary cause of decline is nutrient depletion, as the mycelium consumes all available carbohydrates and proteins within the agar medium. Once the food source is exhausted, the fungus cannot maintain active growth, leading to senescence and eventual death of the culture.
Another major factor is desiccation, or the drying out of the agar medium. Even when sealed, the water content slowly evaporates, leaving the mycelium in a dry, inhospitable environment. Since fungal cells rely on moisture for nutrient absorption, water loss quickly reduces viability and prevents successful regrowth.
As the mycelium grows and metabolizes, it releases waste products into the confined agar environment. The accumulation of these metabolic byproducts can reach toxic levels, inhibiting further growth and contributing to the culture’s degeneration. This buildup of waste causes a decline in the culture’s vigor, often resulting in a loss of desirable genetic traits.
The risk of contamination from airborne molds or bacteria is always present, especially during plate preparation or transfer. A single contaminant can rapidly outcompete and destroy a viable mycelial culture, making aseptic technique a constant requirement for long-term storage.
Techniques for Long-Term Culture Preservation
To extend the life of a mycelial culture beyond the standard refrigerated limit, specific preservation techniques are used to halt or drastically slow metabolic activity. One simple, low-tech method is to store colonized agar wedges in sterile, distilled water (the Castellani method). By submerging the mycelium without a food source, the culture enters a deep state of dormancy, remaining viable for a surprising period, sometimes up to 20 years, without refrigeration.
Another technique involves using agar slants—culture grown in test tubes—covered with a layer of sterile mineral oil. The oil prevents desiccation by sealing the agar surface and limits oxygen exchange, slowing the fungus’s metabolism. Cultures preserved under this overlay can remain viable for several years, with some reports indicating successful revival after more than 30 years.
For cultures that must be kept for years or decades, advanced methods like cryopreservation are necessary. This involves freezing the mycelium, often suspended in a cryoprotectant solution like glycerol, at ultra-low temperatures (-80°C or below -130°C). The glycerol prevents ice crystal formation, which would otherwise damage the fungal cells, effectively stopping all metabolic processes indefinitely.
Regardless of the method chosen, periodic subculturing is necessary. A small piece of the stored culture must be transferred to a fresh agar medium to confirm viability and regenerate the culture for active use.