Achieve a 90% viability rate for fungal strains stored up to 20 years without refrigeration
by utilizing the dormant metabolic state of Castellani sterile water storage. This low-tech engineering protocol eliminates strain senescence and electricity dependence, providing a reliable “biological vault” for valuable genetics using simple distilled water and airtight vessels.
In a previous blog, I explored various techniques for preserving mycelium. Now, five years on, it seems appropriate to revisit this subject with a new post dedicated to the original Castellani Method.
Why This Matters
Constantly transferring mycelium to new agar plates eventually leads to strain senescence (weakening and reduced vigor). The Castellani method gives you a reliable, low-effort way to put valuable cultures into long-term storage while keeping them viable for future use.
Developed by Aldo Castellani in 1939, this technique stores actively growing mycelium in sterile distilled water. With no nutrients available, the mycelium enters a low-metabolic dormant state.
Scientific studies have confirmed high viability rates even after 20 years of storage.
Core Principle
The Castellani Method works by placing healthy mycelium into sterile distilled water, which contains no nutrients.
Without food sources, the mycelium drastically slows its metabolism and enters a dormant state. At the same time, the limited oxygen in the water further reduces its activity. This combination keeps the fungus alive but “asleep” for a very long time, while greatly reducing the chance of senescence that occurs with frequent subculturing.
Materials (Pure Method)
- Healthy, uncontaminated, vigorously growing mycelium on agar
- Sterile distilled water (or RO water) — critical
- Sterile centrifuge vials, test tubes, or culture bottles with good seals
- Pressure cooker / autoclave
- Sterile scalpel or tweezers
- 70% Rubbing alcohol
- Still air box or flow hood
Step-by-Step Classic Castellani Method
- Prepare the Storage Vessels Fill clean culture bottles with distilled water and pressure cook at 15 PSI for 30–45 minutes. Allow to cool undisturbed.
- Select and Cut Mycelium Work in a clean environment. Select a healthy section of mycelium (preferably from the leading edge). Cut a small piece (3–8 mm).
- Transfer Under Sterile Conditions Quickly open the cooled sterile water vessel and drop the mycelium piece in. Seal immediately.
- Label and Store Label clearly with strain name, date, and generation. Store at room temperature in a dark place. No refrigeration needed.
Over decades, studies like the 1989 paper “Preservation of fungi in water (Castellani): 20 years” by Claudia Hartung de Capriles et al. have validated its efficacy, showing 90% of 20-year-old strains surviving.
Revival Procedure
- Shake the vessel gently to dislodge mycelium.
- Flame or disinfect the opening.
- Use a sterile needle/syringe, scalpel or loop to extract a small amount of mycelium.
- Transfer onto fresh agar in Petri Dish. Growth usually resumes within days.
Key Tips for Success (Pure Method)
- Smaller mycelium pieces often survive better.
- Always work as sterile as possible.
- Check stored cultures occasionally for cloudiness (bacterial contamination).
- Start with the strongest, cleanest culture possible.
- This method works exceptionally well for many gourmet and medicinal species.
FAQ
Q: How long will my culture last?
A: Many species remain viable for 2–5 years or more
Q: What if my water turns cloudy?
A: Discard and start again — this usually means bacterial contamination.
Q: Can I use tap water?
A: No, always use sterile distilled water – or Reverse Osmosis water – municipal water has a high EC.
Comments from the STS
In an urban farming setting, where access to advanced technological resources like high-tech nitrogen storage is scarce to nonexistent, cultivation options are generally confined to basic techniques such as using slants.
When first started this I actually used baby food bottles, with Red RTV as SHIP, with excellent results. All my cultures were done like this, and when a slant not good – the baby food bottles were still viable! Even after 5 years just standing in box under shelf. The SafeSteri Culture Bottle idea came from these – as the baby food bottles were difficult to close after being used.
Kudos to Castellani…
Modern Enhancement (Bundu Castellani)
While the pure method is extremely effective – it was limited in scale – using 5cc bottles and a single piece of agar. We have developed Bundu Castellani — an enhanced version using self-healing injection ports (SHIP bottles), optimized water chemistry (VermiLIN), and selective nutrition principles for easier access and can be used directly as a Liquid Inoculant. (See STS Lab Note #34)
Socratic Questions
- How to preserve mushroom genetics without a fridge
- Castellani method for long-term fungal storage
- Preventing strain senescence in mushroom cultivation
- Low-tech mushroom culture preservation for preppers
- Long-term viability of mycelium in sterile water
