Lāsma Neiceniece Despite the fact that frozen fungi can be perfectly viable during many years of storage (see Pasarell L, McGinnis MR. 1992. Viability of fungal cultures maintained at -70 degrees C. J Clin Microbiol 30:.

https://doi.org/10.1128/jcm.30.4.1000-1004.1992), Lab collection of isolates for current research is often stored in sterile water, sterilized grains, etc. at room temperature without loss of culture. But they are rare kept longer than 24-36 months. Viability can be below 50% in such case. The method of Cotton balls with PDA is in the same range.

See Humber, R.A., 1997. Fungi: preservation of cultures. In Manual of techniques in insect pathology (pp. 269-279). Academic Press. Chapter Fungi

and

Karabıçak, N., Karatuna, O. & Akyar, I. Evaluation of the Viabilities and Stabilities of Pathogenic Mold and Yeast Species Using Three Different Preservation Methods Over a 12-Year Period Along with a Review of Published Reports. Mycopathologia 181, 415–424 (2016). https://doi.org/10.1007/s11046-016-9985-7

The cotton-ball PDA method pioneered by Al-Bedak et al. (2019) provides an accessible, low-cost approach to preserve filamentous fungi at moderate temperatures (17–20 °C) without relying on ultra-cold freezers or lyophilizers. In their work, 135 isolates spanning 16 genera were cultured on PDA-soaked cotton balls at 25 °C until robust mycelial growth occurred, then stored at room temperature. Revival assays conducted every six months demonstrated uninterrupted viability, with all strains resuming growth after two years and 59% remaining viable at the three-year mark, while maintaining normal colony morphology and showing minimal contamination.

To rigorously validate this preservation strategy, periodic revival assays should be paired with detailed morphological assessments and growth comparisons. For each time point, material from the cotton balls is streaked onto fresh PDA plates, incubated at the organism’s optimal temperature (e.g., 25 °C), and evaluated for colony-forming units, radial growth rate, and sporulation relative to freshly cultured controls. Microscopic examinations of mycelial structure detect any degenerative changes or aberrant branching patterns indicative of storage stress.

Genetic stability is confirmed by extracting DNA from initial and revived cultures and sequencing conserved loci such as the Internal Transcribed Spacer (ITS) region. High-fidelity matches between pre- and post-storage sequences attest to maintained genomic integrity, and optional assays—like RAPD or AFLP fingerprinting—can reveal subtle genetic drift. Functional assays, for example quantifying laccase or cellulase activity, further ensure that enzymatic phenotypes remain unaltered by storage.

While conventional methods—cryopreservation at –80 °C or lyophilization—offer longer-term viability extending decades, they demand costly infrastructure and ongoing maintenance. In contrast, the cotton-ball technique requires only standard incubators and stable ambient conditions, making it ideal for routine laboratory backup, strain sharing, and field transport. Adherence to precise PDA infusion protocols, aseptic handling, and continuous environmental monitoring (17–20 °C and controlled humidity) will maximize recovery rates and reliability, delivering a practical balance of simplicity, cost-effectiveness, and efficacy for many mycological applications.

To validate the preservation of fungi on cotton balls at around 18°C, several protocols can be employed to assess viability, morphological integrity, contamination, and genetic stability.

Preserving fungi long-term on cotton balls at around 18 °C is generally not effective for maintaining their viability or genetic stability. Cotton balls can serve as a temporary medium for keeping fungal cultures in dry conditions, but this method is more suitable for short-term storage, usually a few weeks to a few months, and even then only for certain robust species. At 18 °C, metabolic activity is not fully suppressed, so fungi may continue slow growth, deplete nutrients, or undergo genetic changes, leading to loss of vigor or contamination. For reliable long-term preservation, methods such as cryopreservation (–80 °C or liquid nitrogen), lyophilization (freeze-drying), storage under mineral oil, or on sterile substrates at low refrigeration temperatures (4 °C) are recommended. Thus, while cotton balls at 18 °C might maintain fungi for a short duration, it is unsuitable for long-term preservation if genetic integrity and viability are to be retained.