Sterile technique is a cornerstone of successful mycology

  Whether you're a professional mycologist or a home mushroom grower. Contamination can ruin your cultures and waste valuable time and resources. This guide will walk you through the essential sterile techniques, best practices, and the types of cleaners to use, ensuring your mycological endeavors are successful and contamination-free.

Why Sterile Technique is Crucial

Fungi, like mushrooms, thrive in environments that are also ideal for bacteria, molds, and other contaminants. Sterile techniques help prevent these unwanted microorganisms from infiltrating and ruining your cultures. Maintaining sterility ensures that your fungi grow healthily and without competition from contaminants.

Best Practices in Sterile Technique

1. Clean Workspace:

   • Preparation: Before starting any work, thoroughly clean your workspace. Use a dedicated area if possible, such as a laminar flow hood or a still air box (SAB).
   • Surfaces: Wipe down all surfaces with a disinfectant, such as a 70% isopropyl alcohol solution, bleach solution, or hydrogen peroxide. Pay special attention to high-touch areas.

2. Personal Hygiene:

   • Handwashing: Wash your hands with soap and water before and during the procedure as necessary. Dry them with a clean, disposable towel.
   • Protective Gear: Wear clean gloves, a lab coat, and, if available, a face mask and hair cover to minimize the shedding of skin cells and hair into your work area.

3. Tools and Equipment:

   • Sterilization: Sterilize all tools (scalpels, tweezers, inoculation loops) by flaming them with an alcohol lamp or Bunsen burner. Allow them to cool before use.
   • Storage: Keep sterilized tools in a clean, covered container until ready for use.

4. Media and Containers:

   • Autoclaving: Sterilize media, petri dishes, and jars using an autoclave or a pressure cooker. Follow the manufacturer's instructions for the appropriate time and pressure.
   • Sealing: Once sterilized, seal media and containers with Parafilm or other suitable wraps to prevent contamination.

5. Working Environment:

   • Airflow Control: Minimize air movement by turning off fans and air conditioning. If using a laminar flow hood, ensure it’s functioning correctly and position your work within the sterile airflow.
   • Minimal Movement: Move slowly and deliberately to avoid stirring up contaminants.

6. Inoculation Process:

   • Flame the Tools: Flame tools before and after touching any cultures. This step is critical to prevent cross-contamination.
   • Quick and Precise: Perform inoculations quickly and precisely to reduce the exposure time of sterile media to the environment.

Understanding How Cleaning Products Work

Isopropyl Alcohol (70%)

Mechanism of Action:

Isopropyl alcohol (IPA) works by denaturing proteins and dissolving lipids, effectively disrupting the cell membranes of bacteria and viruses. The 70% concentration is optimal because the presence of water helps to slow evaporation, increasing contact time and enhancing its effectiveness.

Effectiveness:

• Bacteria: Highly effective against a broad spectrum of bacteria, including Gram-positive and Gram-negative species.
• Molds and Fungi: Effective at killing mold spores and fungal cells on surfaces.
• Viruses: Capable of inactivating enveloped viruses (like influenza and coronaviruses) but less effective against non-enveloped viruses.

Bleach Solution (10%)

Mechanism of Action:

Bleach, or sodium hypochlorite, works by oxidizing and denaturing proteins, which disrupts cellular processes and leads to the death of microorganisms. The oxidative properties also break down the cell walls and membranes.

Effectiveness:

• Bacteria: Broad-spectrum bactericidal activity, effective against both Gram-positive and Gram-negative bacteria.
• Molds and Fungi: Very effective against mold spores and fungal cells, capable of penetrating and destroying cell walls.
• Viruses: Effective against a wide range of viruses, including both enveloped and non-enveloped types.

Hydrogen Peroxide (3-6%)

Mechanism of Action:

Hydrogen peroxide acts as an oxidizing agent, producing free radicals that damage cell membranes, DNA, and other essential cellular components. It decomposes into water and oxygen, making it environmentally friendly.

Effectiveness:

• Bacteria: Effective against a broad range of bacteria, including those that form biofilms.
• Molds and Fungi: Capable of killing mold spores and fungal cells through oxidation.
• Viruses: Effective against many viruses, particularly non-enveloped viruses that are resistant to other disinfectants.

Autoclave/Pressure Cooker

Mechanism of Action:

Autoclaves and pressure cookers use high-pressure steam to achieve sterilization. The combination of heat (usually around 121°C or 250°F) and pressure (typically 15 psi) denatures proteins and disrupts cellular structures, leading to the death of all microorganisms, including resistant spores.

Effectiveness:

• Bacteria: Sterilizes all forms of bacteria, including spore-forming species.
• Molds and Fungi: Effective against fungal spores and mycelium.
• Viruses: Kills all viruses, including highly resistant ones.

Flame Sterilization

Mechanism of Action:

Flame sterilization involves exposing tools to direct flame, usually from an alcohol lamp or Bunsen burner. The high temperatures (above 1000°C or 1832°F) rapidly denature proteins and nucleic acids, causing instant cell death.

Effectiveness:

• Bacteria: Instantly kills all bacteria, including spores.
• Molds and Fungi: Destroys all fungal cells and spores on contact.
• Viruses: Kills all viruses by denaturing their genetic material and proteins.

UV Sterilization

Mechanism of Action:

Ultraviolet (UV) light, particularly UV-C (200-280 nm), damages the DNA and RNA of microorganisms through the formation of thymine dimers, preventing replication and leading to cell death.

Effectiveness:

• Bacteria: Effective against a wide range of bacteria, though some may require longer exposure times.
• Molds and Fungi: Can kill mold spores and fungal cells on surfaces and in the air.
• Viruses: Effective against many viruses, including both enveloped and non-enveloped types.

Step-by-Step Sterilization Process for a Home Lab

1. Setting Up:

   • Clean and disinfect your workspace with a 70% isopropyl alcohol solution.
   • Set up your still air box or laminar flow hood if available.

2. Preparing Media:

   • Mix your growth media according to instructions.
   • Pour the media into containers (e.g., petri dishes or jars) and cover loosely.

3. Sterilizing Media:

   • Place the media containers in an autoclave or pressure cooker.
   • Sterilize at 15 psi for 15min - 2.5hrs (adjust according to your media’s requirements).

4. Inoculation:

   • Wash your hands and wear gloves.
   • Flame sterilize your inoculation tools.
   • Transfer your cultures quickly and precisely.
   • Seal the inoculated media with Parafilm or other suitable materials.

5. Incubation:

   • Place your inoculated media in a clean, controlled environment.
   • Monitor for contamination regularly and dispose of contaminated cultures safely.

By following these sterile techniques and best practices, you'll create an environment conducive to successful mushroom cultivation. Cleanliness, precision, and the right tools and cleaners are your allies in the fight against contamination. Happy growing!