The cultivation of mycelium, the vegetative body of a fungus, often begins on agar plates, which serve as a nutrient-rich, solid growth medium. Once a plate is fully colonized by a healthy, uncontaminated culture, appropriate storage is necessary to maintain the organism’s genetic integrity and viability. Preservation methods slow the mycelium’s metabolic rate, preventing it from exhausting its food source and drying out, which leads to senescence or death. Successfully storing these cultures is fundamental for preserving specific strains for future inoculation projects.
Preparing Agar Plates for Storage
Before placing an agar plate into storage, a thorough physical examination must confirm the culture’s purity. A visual inspection checks for signs of contamination, such as bacterial films, distinct mold colonies, or discoloration. Only plates exhibiting clean, vigorous mycelial growth should be selected, as storing a contaminated plate preserves the contaminant.
Once a clean culture is confirmed, the plate must be properly sealed to prevent dehydration and the entry of airborne microbes. Specialized laboratory film, such as Parafilm, is the preferred material due to its semi-permeable nature. This film creates a tight seal around the perimeter, significantly reducing moisture loss while still allowing the necessary exchange of gases like oxygen and carbon dioxide. This gas exchange supports the mycelium’s slow aerobic metabolism during storage.
The final preparation involves clear and permanent labeling of the culture plate. Mandatory information should include the full strain name or genetic identifier, the date the culture was transferred, and the specific type of agar medium used (e.g., Potato Dextrose Agar or Malt Extract Agar). This detailed labeling ensures accurate tracking and retrieval, eliminating confusion about the culture’s age and identity when it is needed later.
Short-Term Storage Methods
Mycelium cultures intended for use within three months can be maintained using less intensive, short-term storage methods. The primary goal is to slow the growth rate sufficiently to avoid premature exhaustion of the agar’s nutrients. This is achieved by finding a suitable location protected from extreme environmental factors.
Optimal placement involves storing the sealed plates in a dark cabinet or on a dedicated shelf away from direct sunlight or heat, which would accelerate growth and moisture evaporation. The ideal ambient temperature is slightly cooler than the strain’s optimal colonization temperature, typically ranging from 60–70°F (15–21°C). This temperature range slows mycelial expansion, allowing the plate to remain viable without requiring immediate subculturing.
At this temperature, the mycelium remains metabolically active, but its growth is significantly reduced. The culture remains healthy for approximately three months by consuming nutrients at a diminished rate. Beyond this timeframe, the culture risks consuming too much media or experiencing desiccation, necessitating a move to a longer-term preservation technique.
Long-Term Preservation Techniques
For archival purposes, extending viability from six months up to several years requires rigorous preservation techniques to dramatically slow mycelial metabolism. The most common and effective method for long-term preservation is refrigeration, which uses cold temperature to induce a state of near-dormancy.
The ideal storage temperature for refrigerated cultures is a consistent 35–40°F (2–4°C), just above freezing. This low temperature significantly depresses the enzymatic activity within the fungal cells, effectively pausing the mycelium’s growth and metabolic processes. Slowing the rate of nutrient consumption and waste product accumulation extends the life of the culture substantially beyond what is possible at room temperature.
Refrigeration environments are dry, posing a significant threat of desiccation to the agar medium. Therefore, the sealing of the plates must be impeccable, using multiple layers of specialized film or wrapping the sealed plate in an additional layer of plastic wrap or a zip-top bag. This superior sealing creates a secondary moisture barrier, preventing the agar from drying out, which is the most common cause of failure in refrigerated storage.
Advanced Archival Techniques
While refrigerated agar plates are convenient, more advanced archival techniques exist for greater longevity. Agar slants, which are test tubes containing agar solidified at an angle, offer greater surface area and a reduced rate of evaporation compared to flat plates. Slants often extend viability to a year or more. Overlaying a slant culture with sterile mineral oil or liquid paraffin creates an oxygen-deprived barrier that can preserve cultures for several years by limiting gas exchange and preventing desiccation.
Assessing and Revitalizing Stored Cultures
When a mycelium culture is removed from long-term cold storage, it must be handled carefully to ensure a successful return to active growth. Refrigerated cultures require a period of acclimation to prevent thermal shock. The sealed plate should be allowed to warm up slowly to room temperature over several hours before being opened, allowing the mycelium to gently resume its normal metabolic functions.
Following acclimation, a careful visual assessment is mandatory to check for contamination and senescence. Signs of mold, bacteria, or a wet appearance must be noted, as these compromise the culture. Senescence, or aging, is often indicated by thin, wispy, or distorted mycelial growth, suggesting the culture is weakened or genetically degraded from stress.
To ensure viability before use, revitalization through subculturing is necessary. This involves carefully transferring a small, healthy piece of the stored mycelium to a fresh, nutrient-rich agar plate. This transfer provides a new food source, allowing the mycelium to resume vigorous growth and confirming the genetic line remains healthy.