Mycology involves transferring microscopic spores into a nutrient-rich material called a substrate to initiate fungal growth. This inoculation is commonly achieved using a sterile spore syringe, which contains millions of mushroom spores suspended in purified water. The spore syringe holds dormant genetic material, unlike a liquid culture syringe which holds actively growing mycelium. Success depends on executing the methodology with precise care to prevent contamination. Users must ensure compliance with all local laws and regulations, as the legality of cultivating certain fungal species varies significantly by region.
Essential Preparation and Materials
Before beginning the injection process, gathering and preparing the correct materials is necessary for a sterile procedure. The primary components include the spore syringe, typically 10-12 cc, and the prepared target substrate. The substrate must be ready, such as a sterilized grain jar, an all-in-one substrate bag with a filter patch, or an agar plate.
Required Materials
To maintain a clean environment, several items are required:
- Disposable gloves and a face mask to minimize airborne contaminants.
- 70% isopropyl alcohol wipes or solution for sanitizing surfaces and equipment.
- A small flame source, such as a butane torch or lighter, to sterilize the needle tip.
Maintaining a Sterile Environment
Stringent aseptic technique is required because competing microorganisms like molds and bacteria are the most frequent cause of failure. A dedicated workspace must be established, often utilizing a Still Air Box (SAB). The SAB is a simple plastic enclosure that minimizes air movement and turbulence. Working within this still air zone ensures that airborne contaminants quickly settle rather than circulating onto sterile materials. The workspace, including the inside of the SAB, must be thoroughly wiped down with 70% isopropyl alcohol before use. The user should wear gloves and a mask, and gloved hands must be sterilized with alcohol before entering the work zone. The SAB is a highly effective and budget-friendly alternative to a laminar flow hood for the hobbyist.
Step-by-Step Spore Injection Procedure
Syringe Preparation and Sterilization
Prepare the spore syringe by gently shaking it to break up any spore clumps and evenly distribute the material throughout the sterile water. Before the needle penetrates the substrate, sterilize the metal tip using the flame source until it glows red hot. This incinerates all microbial life. Allow the needle to cool for 15 to 30 seconds before proceeding. Injecting with a hot needle will destroy the spores and may melt the substrate container’s plastic components.
Injection Technique
Insert the needle swiftly through the self-healing injection port or a small, sanitized area of the substrate bag. Dispense the spore solution by slowly depressing the plunger, typically injecting between 0.5 cc and 2 cc of liquid per injection site, depending on the substrate volume. Using multiple injection points (three or four) helps distribute the spores and encourages faster colonization. Once the solution is dispensed, withdraw the needle. If the container lacks a self-healing port, immediately seal the injection site with a small piece of tape. Flame-sterilize the needle again after each injection if inoculating multiple containers.
Post-Injection Incubation and Observation
Incubation Conditions
The inoculated substrate requires a specific environment to encourage spore germination and mycelial colonization. Place the container in a clean, dark area with a consistent ambient temperature, as light is not required during this initial growth phase. The optimal temperature range for colonization is generally between 20°C and 25°C (68°F to 77°F). Maintaining a steady temperature is important; low temperatures slow colonization, while temperatures exceeding 25°C increase the risk of contamination.
Monitoring Growth and Contamination
Within two to six weeks, initial signs of mycelial growth should become visible. This appears as a dense, white, thread-like or cottony network spreading across the substrate as the healthy mycelium consumes nutrients. The grower must closely monitor the substrate for any signs of contamination, which indicates a failure in aseptic technique. Common contaminants appear as patches of color other than white, such as green (Trichoderma mold), black, or grey. A slimy appearance or a sour, unpleasant odor are strong indicators of bacterial contamination. If contamination is observed, the material should be discarded immediately to prevent the release of spores that could compromise future cultivation attempts.