Chapter 2: Sterile Technique/Tek & Agar Culture

High Detail Review Video of The Mushroom Cultivator by Paul Stamets and J.S. Chilton

Mycological and marketing expert Benjamin Ashpole explores methods for creating a clean workspace and reducing contamination—essential for successful mushroom cultivation. Discover how to protect your mycelium from competing microorganisms, whether in a closet or lab.

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Chapter 2: Sterile Technique/Tek & Agar Culture

Find key topics:

  • Clean Workspace: Convert any space into an inoculation chamber with basic tools like a torch and bleach.

  • Fight Contamination: Minimize the five main sources of contamination using HEPA filters and other methods.

  • Modern Methods: Use a plastic tote for a mini clean room and sterilize tools for a semi-sterile environment

Conquer Contamination and Boost Your Mushroom Yields: Mastering Sterile Technique for Agar Culture

Delving into the world of home mushroom cultivation reveals that achieving success hinges on many factors, with sterile technique standing out as absolutely crucial. Chapter 2 of "The Mushroom Cultivator: A Practical Guide to Growing Mushrooms at Home" by Paul Stamets and J.S. Chilton meticulously outlines the principles of this vital practice, and a thorough understanding of its concepts can dramatically increase your chances of a successful mushroom harvest.

The Importance of a Clean Working Environment

The book clarifies that while true "sterility," meaning the complete absence of all microorganisms, is exceptionally difficult to achieve, especially for home growers, establishing a significantly cleaner or highly sanitary environment is often sufficient for successful cultivation endeavors. Many aspiring mycologists initiate their work in accessible spaces like a spare closet, a guest bedroom, a basement corner, or even a partitioned-off section of a room.

The core objective is to designate a specific area that can be maintained with a high degree of cleanliness, thereby minimizing the potential for competition from unwanted bacteria, yeasts, and other fungal spores that can contaminate your intended mushroom cultures.

Understanding Sources of Contamination

As the book aptly points out, the air around us is a dynamic environment filled with microscopic life, including fungi, bacteria, and viruses, easily carried by even the slightest air currents. These ubiquitous microscopic particles pose a constant challenge to maintaining clean cultures. To effectively manage this, the book identifies five key sources of contamination in the context of mushroom cultivation:

  • The immediate external environment

  • The culture medium (the material on which the fungus is growing)

  • The culturing equipment

  • The cultivator and their clothing

  • The mushroom spores or mycelium being cultivated.

Constructing Your Clean Lab Space

To effectively address these contamination sources, the initial step often involves creating an inoculation chamber or a dedicated clean laboratory space. While commercially produced clean chambers are now readily available for purchase, the book provides detailed instructions on how to transform a small existing room, such as a walk-in closet or a pantry, into a functional inoculation area. This process involves:

  1. Removing all items that can harbor dust and spores, such as rugs, curtains, and other fabric-like materials.

  2. Thoroughly cleaning all surfaces – floors, walls, and ceiling – with a mild disinfectant. Painting the room with a high-gloss white enamel is recommended to simplify future cleaning.

  3. Sealing any windows or other potential sources of air leaks with plastic sheeting.

  4. Constructing an anti-chamber or airlock at the room's entrance using plastic sheeting or other materials. This airlock acts as a protective barrier, ensuring the main clean room door remains closed while entering or exiting.

Whether opting for a dedicated room or a more compact solution like a plastic tote, equipping your workspace with the appropriate tools is paramount for successful sterile work. The book advises having the following items readily available within the lab space:

  • A clean chair and a sturdy table with a smooth, easily disinfected surface

  • A propane torch, alcohol lamp, Bunsen burner, or even a butane lighter. It's crucial to recognize that the primary function of this heat source is to sterilize tools like tweezers and scalpels used for tissue and agar transfers, not to sterilize the air.

  • Clearly labeled spray bottles containing a 10% bleach solution and/or 70% isopropyl alcohol for surface disinfection. Peroxide is also mentioned as a possible cleaning agent. The book cautions about potential corrosion from bleach on certain metals, suggesting the use of appropriate materials or alternative solutions like isopropyl alcohol, which, while not as potent as bleach, offers a less corrosive option. Even diluted bleach solutions can be effective with extended contact times

  • Sterile Petri dishes and test tube slants. Test tube slants are noted as a more advanced tool suitable for longer-term culture storage

  • Stick-on labels, a notebook, a ballpoint pen, and a permanent marker for detailed record-keeping. Maintaining accurate notes is emphasized as a critical aspect of successful cultivation

  • An agar knife and an inoculation loop, which can be a scalpel or another sharp knife.

Maintaining a Strict Hygiene Regimen

Once your sterile work area is established, adhering to a strict and unwavering cleaning routine is non-negotiable. This includes consistently cleaning with disinfectants, mopping the floors, and even air washing with a fine mist of a 10% bleach solution. It is vital to ensure proper ventilation and to refrain from re-entering the laboratory for at least 15 minutes after spraying to allow suspended particles to settle. A fundamental principle highlighted is that preventing contamination is significantly easier than trying to eliminate it after it has occurred.

Addressing Persistent Contamination

For cultivators who continue to experience high rates of contamination despite their best efforts, the book suggests several additional strategies. These include:

  • Utilizing a colloidal suspension of light oil in the laboratory's atmosphere to trap airborne contaminant particles

  • Constructing a still air chamber, often referred to as a glove box or, more commonly now, a plastic tote with armholes, to provide a semi-sterile environment with minimal air movement for conducting transfers. The key benefit of a still air chamber is the reduction of air currents, allowing airborne contaminants to settle onto disinfected surfaces

  • Employing high-efficiency particulate air (HEPA) filters to remove airborne contaminants. Modern, affordable HEPA filters designed for desktops or portable use can significantly improve air quality in small spaces through constant recirculation and filtration. In more advanced setups, laminar flow hoods with integrated HEPA filters create a positive pressure sterile environment.

Whether you are a beginner venturing into the fascinating world of mushroom cultivation or an experienced grower aiming to optimize your yields, a thorough understanding and diligent application of sterile technique, as comprehensively outlined in "The Mushroom Cultivator," is a cornerstone of success. By prioritizing cleanliness, minimizing airborne contaminants, and consistently adhering to a strict hygiene protocol, you can substantially mitigate the risks of contamination and consistently cultivate healthy, high-yielding mushroom cultures

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