Chapter 2: Starting a Culture from Spores
High Detail Review Video of The Mushroom Cultivator by Paul Stamets and J.S. Chilton
Mycological and marketing expert Benjamin Ashpole discusses how to obtain pure spore prints and start a multi-spore culture. Learn how to sterilize tools, germinate dehydrated spores, and isolate mushroom mycelia to establish a pure culture free of contamination.
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Chapter 2: Starting a Culture from Spores
Find key topics:
Spore Collection: Learn how to collect nearly contaminant-free spores using the mushroom's natural veil
Germination Techniques: Discover methods for germinating spores, including using sterile water and inoculating multiple media dishes to improve success
Pure Culture Isolation: Understand how to selectively isolate mushroom mycelia and prevent contamination to establish a pure culture
Starting Your Mushroom Culture from Spores
Mushroom cultivation often begins with the fascinating process of initiating a culture. "The Mushroom Cultivator: A Practical Guide to Growing Mushrooms at Home" by Paul Stamets and J.S. Chilton is a foundational text for both novice and experienced growers, offering detailed methodologies for every stage of cultivation. Here, the focus is on the techniques for starting a culture from spores.
Historically, the reliable cultivation of mushrooms was a closely guarded secret. The publication of "The Mushroom Cultivator" in 1983 marked a significant shift, aiming to empower new growers to cultivate a wide array of mushroom species. At the time, resources for beginner growers were limited, making self-sufficiency a valuable skill. While modern growers benefit from readily available quick-start options, understanding the fundamentals outlined in this book remains invaluable for long-term success and a deeper understanding of fungal biology.
There are primarily two methods for initiating a mushroom culture: starting from spores or cloning live tissue. While cloning involves taking a piece of the interior of a fresh mushroom, establishing a healthy clone becomes increasingly challenging a day or two after the mushroom has been picked. This exploration will focus on the method most often embraced by beginners: starting a culture from spores.
The advantage of using spores is their longevity; they can remain viable for weeks to months after the mushroom has decomposed.
Step-by-Step Guide to Collecting Spores: Taking a Spore Print
The first step in this process is collecting spores by taking a spore print. To do this, the cap of a fresh, clean mushroom is severed from its stem and placed gills-down on a non-porous surface such as white paper, clean glass (like a microscope slide), aluminum foil, or even black paper for better contrast depending on the spore color. Mushroom spores exhibit a diverse range of colors, including white, brown, purple, and black. If the mushroom specimen is partially dried, adding a drop or two of water to the cap's surface can aid in spore release. To prevent evaporation and dispersal by air currents, a cup or glass is often placed over the mushroom cap. After several hours, the spores will have fallen in a pattern reflecting the radiating symmetry of the gills.
On Paper: If the spore print is taken on paper, it can be cut out, folded in half, sealed in an airtight container, and labeled with the date, species, and collection number
On a Microscope Slide: When using a microscope slide, the spores can be sandwiched between two pieces of glass and taped along the edges to prevent contamination
In a Petri Dish: For enhanced sterility, collecting spores on a clean, heat-sterilized petri dish by placing the cap over the edges (without the gills touching the surface) is also a recommended practice. This method minimizes the risk of collecting live tissue or bacteria, which could lead to contamination or a clone instead of a new generation from spores. Once collected in a petri dish, the lid can be replaced to seal it.
Initiating the Culture: Sterilization and Inoculation
Once a spore print is obtained, the process of mushroom culture can begin with sterilization. An inoculating loop or scalpel should be sterilized by holding it over the flame of an alcohol lamp, butane torch, candle, or another heating device. Induction heaters are also noted as a potentially convenient alternative to open flames, especially when working near flammable substances like alcohol. After heating, the tip of the tool should be cooled by inserting it into the sterile media in a petri dish. Then, some spores are carefully scraped off the print and transferred by streaking the tip of the transfer tool across the agar surface. Various tools can be used for this transfer, including scalpels or tools with a small wire loop at the end. Another method involves scraping the spores above an open petri dish and allowing them to fall onto the medium.
Increasing Success: Inoculating Multiple Dishes
When starting a new culture from spores, it is advisable to inoculate at least three media dishes to increase the likelihood of successful germination. Mycelium grown from spores in this manner is termed a multi-spore culture. The rationale behind using multiple dishes is that some spores might not germinate, or the process might take varying amounts of time. It's often a good practice to both attempt immediate cultivation on a nutrient-rich medium like agar and store some spores for later use, effectively building a mycelium or mushroom library as a backup.
Viability of Spores and Hydration Technique
Freshly produced spores are typically moist and inflated cells with a high rate of germination. Over time, they tend to dry, collapse, and become more difficult to germinate. The probability of germinating dehydrated spores can be increased by hydrating them in sterile water for 6 to 12 hours after a brief sterilization period. This spore solution can then be used to inoculate several agar plates with a few drops each. However, it's crucial to remember that if the original spore print was taken under unsanitary conditions, this technique could equally favor the growth of contaminant spores.
Observing Germination and Identifying Contamination
With either direct spore transfer or spore solution inoculation, spore germination and initial signs of contamination should become evident within three to seven days, although allowing at least a week or two can make it easier to distinguish contaminants from developing mycelium, especially for beginners. The mycelial strands typically appear grayish and diffuse initially, gradually becoming whitish as they divide, grow, and spread through the medium. It's important to note that the color of the mycelium can vary depending on the mushroom species, with many beginner-friendly species exhibiting fluffy white mycelium. Mycelial growth should originate from a central point as threadlike strands.
Isolating Pure Cultures
Once mushroom mycelium has been identified, sites of germinating spores should be transferred to new media dishes. This selective isolation allows the cultivator to establish a pure culture free of contamination. If contamination appears concurrently with mycelial growth, segments of the emerging mycelia can be carefully cut away from the contaminant colonies and transferred to a fresh dish. Precautions should be taken to avoid disturbing sporulating molds to prevent the spread of their spores, and the scalpel should be cool before cutting into the agar to prevent an explosive release of spores from neighboring molds.
Understanding Multispore Cultures
Multispore culture is considered a less difficult, yet reasonably efficient, method for obtaining a viable culture, even if it's not absolutely pure. The germination of numerous spores results in the creation of many different strains, some of which may be incompatible with others or exhibit variations in their fruiting behavior under artificial conditions. This mixture of strains can sometimes limit overall yields, as less productive strains might hinder the activity of more productive ones. However, strains derived from domesticated parents that fruit well in laboratory conditions have a higher likelihood of their progeny behaving similarly.
Cultures originating from wild specimens might initially fruit poorly in an artificial environment, necessitating selective development, akin to cultivating wild plants. These wild strains can possess unique characteristics such as different flavors, shapes, colors, or textures, making their cultivation worthwhile. Among the diverse strains resulting from multispore germination, some may only be capable of vegetative growth, meaning they can absorb nutrients but not produce mushroom fruiting bodies. A monocaryon, which is a network of cells originating from a single spore, typically cannot produce fertile, spore-bearing mushrooms. However, when two compatible monocaryons meet and mate, they exchange genetic material, resulting in dicaryotic mycelium, which can produce fertile offspring in the form of mushrooms.
Advanced Techniques: Single Spore Isolates
For cultivators primarily interested in obtaining a viable culture, multispore germination generally suffices. However, for those interested in crossing monocaryotic strains and studying mating characteristics, the technique of single spore isolates is employed. This involves diluting spores in sterile water multiple times to obtain a very low concentration, which is then used to inoculate agar dishes, allowing for the isolation of individual monocaryons.
The Challenge of Contamination
A significant challenge in concentrated multi-spore germinations is the increased risk of contamination, particularly from bacteria. Some bacteria can parasitize the mycelial cell walls, while others stimulate spore germination only to later digest the resulting mycelium. Conversely, certain fungi have developed symbiotic relationships with other microorganisms, where some bacteria and yeast can actually stimulate spore germination in mushroom species that are otherwise difficult to grow in sterile culture. For example, the spores of Chanterelles do not typically germinate under artificial conditions until specific microorganisms and activated charcoal are added to the media. While some bacteria can be beneficial, most contaminants encountered in mushroom cultivation are detrimental. Maintaining a diligent regimen of hygiene, using HEPA filters, and employing good laboratory techniques are crucial for minimizing these costly contaminants.
While contemporary growers have access to easier, albeit potentially more expensive, quick-start options like pre-inoculated substrates, understanding the process of starting a culture from spores, as detailed in "The Mushroom Cultivator," provides a fundamental knowledge base for any aspiring mushroom grower. This method, though requiring more patience and attention to detail, unlocks a vast potential for cultivating diverse mushroom species and deepening one's appreciation for the fascinating world of fungi.
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