Hey all,
I recently got a request from another member to do a deep dive on isolating cultures from culinary mushrooms. I have worked in the culinary mushroom industry for many years in the production and development of commercial spawn products. The facilities I managed produced several tons of spawn and ready to fruit products per day and I have done additional consulting work in the industry since then. Many of the methods from the industrial sector never trickles down to the hobbyist sector so feel like I might be able to provide some worthwhile information or at least a different perspective.
This is going to be rather long brain dump from my time in the industry and cover a couple different topics. I’m going to try to break them out into easy to find sections. Fair warning, there may be some repeat information to help pad the edges for those who are looking for topics only.
I will do my best to cover industry standards as well as more hobbyist-tangible and economical alternatives. Many of the methods that I am going to recommend come from my personal preference over the years. They will focus on producing the most consistent and reliable end products while mitigating as many risks as possible. I am certain that many of my points will be in direct opposition of those in the hobbyist scene, but I will do my best to explain the reasons why I recommend one method over the other. As discussion progresses, I will be editing this document to include common questions and to improve clarity.
Aseptic/sterile technique
These terms are used more or less interchangeably, with the former seeming to be coming into fashion over the latter. I will try to remain consistent but for the purposes of this guide their meanings are identical.
Aseptic technique is the method by which a technician prevents contaminating a medium, substrate, or culture with unintended microorganisms. This is the most important skill you can develop while beginning your journey into mycology. Sources of contamination are endless. Unless properly treated, each surface, tool, piece of clothing, and puff of air, is teeming with microbial life that will inevitably be better suited to the substrate you are using than your intended culture.
Decontamination
Cleaning vs disinfection vs pasteurization vs sterilization
In order to prepare an environment that is conducive to culture work, we need to know the difference between the above terms. Many people when they start out, think that a few quick sprays of alcohol is enough to give them a sterile environment to work with. While you can get away with that for a little while, for continued success you will need to ultimately understand and perform most of the above.
Cleaning aims to remove any soils or films on a surface. If these are left on a surface during disinfection or sterilization, the microorganisms in the air will begin to recolonize them. If the films or soils are too deep, they can also harbor contaminants which will continue to grow and threaten your work.
For our purposes, water will be the primary cleaning agent. A neutral, non-antimicrobial, detergent that is free from colors and fragrances can be used but be sure to rinse with copious amounts of water to ensure there are no films left behind.
Disinfecting aims to kill all the microorganisms still living on a surface. These methods often won’t penetrate deeply into the material which is why it is important to clean first. These methods are often all that can be done for surfaces and tools that cannot stand heat or harsh chemicals. Additionally, disinfection methods may be used in conjunction with sterilization methods both before and during culture work.
Disinfection methods can involve chemicals such as 70% ethanol, 70% isopropanol, household bleach, and 35% hydrogen peroxide, or other methods such as open flame. We will discuss these as they become relevant. For most technicians, a form of 70% alcohol for surfaces is sufficient. For tools, an alcohol an alcohol lamp and a smaller container of 70% alcohol is sufficient. Before working with alcohol and open flame, please see the “Safety” section.
Pasteurization seeks to kill enough of the present load of microorganisms and their spore producing structure in order to slow their growth. This allows the pasteurized material to resist spoilage for a longer period of time, and also allows subsequently added microorganisms the ability to outcompete the local flora.
Sterilizing goes one step further and aims to destroy spore structures that would otherwise resist disinfection. While sterilization is a powerful tool, it is only as good as the environment in which it resides and the technique of the technician. The main method of sterilization that we will focus on is steam sterilization.
As a matter of practice, be sure to thoroughly clean before attempting any of the other decontamination methods.
The final item on the topic of decontamination will be related to the air. Air is not a surface and therefor cannot be sterilized in conventional ways yet it is arguably one of the more important aspects of mycology.
Laminar Flow Hood
Filtering the air is the most effective way to ensure that it doesn’t contaminate your newly sanitized work environment and cultures. Unfortunately, standard air filters and fans are not enough to guarantee clean air. To ensure a proper working environment with filtered air, a laminar flow hood will be required. Laminar flow hoods use a strong fan, to blow out a continuous solid wall of decontaminated air. When built correctly, this prevents any unclean air from reaching your work surface. I won’t be including instructions on how to build one, but there are a number of different designs out there. Search laminar flow hood and you’ll find tons of schematics. My personal design preference is for a unit that blows air directly at the technician, not onto the work surface. I’ll explain why in just a bit.
Using a laminar flow hood
Before starting culture work, check the pre-filter and ensure that it is relatively clean. Using prefilters will dramatically extend the life of the HEPA filter and will delay its inevitable costly replacement. Next, turn on the laminar flow hood and begin cleaning and sanitizing the working area including the walls and “ceiling” of the hood. Be sure to avoid getting the filter wet with any water or reagents. Now you can begin setting up the equipment you will be using for the day. Ensure that all items are clean, and sanitize them within the filtered air path of the laminar flow hood. Resanitize your work surface if necessary. Allow the blower to run for another 5 minutes, during this time wash your hands and sanitize them with 70% alcohol. You can also spray down the front of your clothes with alcohol as well.
While working in the laminar flow hood, be mindful that there is a gradient of cleanliness that extends perpendicularly from the plane of the filter. As the air touches more objects and surfaces, the likelihood of it getting contaminated by something increases. So, if your filter is blowing air towards you, place the cleanest or most sensitive materials towards the back and do you best to work from the cleanest to the dirtiest areas. This becomes especially important when working with fresh mushrooms as well as sporulating and contaminated cultures. Any of those items will create spores that will contaminate everything down wind. If your laminar flow hood blows from the top down, you unfortunately don’t have that level of control and spores will be spread everywhere.
Still air box
An alternative to the laminar flow hood is the still air box. A still air box is a clear box that attempts to reduce the amount of contaminated airflow across the work surface. These units can be made quite cheaply from common household items and they do work. That said, they are much more difficult to use and have some serious limitations.
A common design for a still air box is a clear plastic storage tote with two holes cut into it that are big enough for the technicians arms to fit through. Prior to work, the inside of the box is sprayed thoroughly with alcohol and then sealed. This allows the alcohol to evaporate and essentially disinfect the air. Obviously one this opens up again, air can still get in but the goal isn’t a totally clean work surface, just a cleaner one. From here though, it is easy to see how this can be limiting. No open flames can be used inside the box without cutting additional holes in the plastic, any new holes or removal of tools generate new air currents, and your working space is severely limited. For someone serious about the hobby, please consider investing in a laminar flow hood.
Media preparation and theory
Media preparation methods and materials are going to vary greatly depending on the items you have available to you. Because of this, there are an infinite number of recipients and there is no perfect mix. That said, there are some general principles one can employ that can help you craft better media.
Agar vs Liquid culture
This is going to be a slightly controversial section. I am not a proponent of liquid culture for a number of reason. It certainly has its merits but in my opinion, when given a choice, agar is the superior method of culture.
Agar
Agar culture involves some kind of nutrient broth or solid nutrient mix and a gelling agent that when hot, can be poured into just about any heat safe container. Typically large batches of media are mixed up, sterilized, and aseptically poured into petri plates or slanted tubes. Mycelium or spores are then aseptically transferred onto the surface of the agar and then grow out in a roughly lateral manner.
From there, sections of new actively growing mycelium are then cut away from the agar and aseptically transferred to new medium to continue growing.
Liquid culture
In liquid culture a nutrient broth solution is made and sterilized without any gelling agent and mycelium or spores are added directly to the broth. Agitation or other methods of oxygenation are recommended as the oxygen dissolved in the broth will generally not be sufficient. In this type of medium, the mycelium will grow in a roughly spherical shape and will begin to clump together as the liquid is agitated.
Once fully grown, the liquid is then agitated further to break up the mycelium for ease of inoculation. The liquid is either then aseptically filtered and added to a low nutrition suspension, or more commonly the liquid containing fragments of mycelium is removed via syringe or pump and then injected into bulk substrate.
This offers a number of advantages. Liquid is easy to prepare, costs much less than agar, transfers using only a syringe, and doesn’t require the use of an aseptic environment or equipment other than some alcohol when inoculating bulk substrate. This is especially convenient for hobbyist growers. Additionally, spores can be suspended in neutral buffer and shipped without legal issues for active varieties while also offering ease of inoculation.
That having been said, I’m going to try to make my case for converting to agar work if you are seriously considering the hobby. While agar does require more attention, materials, equipment, and skill, it provides you with way more flexibility, information, and will improve quality of your cultures.
One of the biggest advantages of using agar is the ability to actively monitor and manage the health of your culture. Based on the type of strain and the type of media used, the morphological characteristics of the mycelium can tell you a lot about its health. Additionally, with agar culture you have the opportunity to select specific morphological expressions from the mycelium to ensure a consistent, even culture. Furthermore, should a culture become contaminated, you can not only identify the contaminant, but you also have a decent chance of rescuing the culture itself. In liquid culture, you have no real way of doing any of this.
Bulk inoculum
Bulk inoculum refers to the intermediate stage between the culture maintenance stage and the fruiting stage. This is where technicians transfer small pieces of their cultures to larger amounts of media for the purpose of making large amounts of inoculum. This step is going to vary for each individual depending on their end goal. For some, will this could mean making more liquid culture, agar plates, grain flasks, or even a more tailored bulk substrate in 25lb (11kg) bags. All the techniques discussed so far will continue to remain important. Each transfer from vessel to vessel will still require impeccable aseptic technique and a suitable work environment. I will go into more detail for specific substrates in the “Optimizing your substrate” section.
A few parameters that do get added to the mix are substrate temperature, air temperature, ambient CO2 levels, and gas exchange. Fungi, sharing many similarities with animals, generate large amounts of heat and carbon dioxide. As vessels increase in size, so does the amount of biomass, and in turn, so does the amount of carbon dioxide and heat. These principles do not stop being relevant during fruiting.
Temperature control
Once vessels reach about 2lbs (~1kg) in size, heat becomes a problem. A nutritionally optimized substrate generally allows for rapid expansion of mycelium and in turn, causes a rapid spike in heat. This heat, if left unchecked, can reach temperatures that can retard growth and even kill sections of mycelium. While many people use air temperature to monitor their growing environment, substrate temperature is a better indicator of the health of the fungi. For example, oyster mushrooms growing in 25lb (~11kg) bags pure alfalfa can reach over 115F (~46C) in a 34F (~1C) cooler. For most culinary mushrooms, substrate temperature over 80F (~26C) is where damage and yield loss begins to occur.
Gas exchange
Gas exchange is the other parameter that must be controlled for as the amount of biomass grows. This can become a problem even before bulk inoculation if there are a large number of smaller cultures all in a poorly ventilated room or refrigerator. As the fungi begin to break down their substrate, they release large amounts of carbon dioxide, just like animals. Like all animals, most fungi need oxygen in order to continue respiration. Fungi also use carbon dioxide concentrations in the air to signal various parts of their life cycle.
Fortunately, most containers designed for fungal culture have filter patches that will properly regulate airflow and gas exchange. That said, when using canning jars, care must be taken to include a properly sized filter patch.
One final note on carbon dioxide, CO2 is a heavy gas which means when there are no air currents to mix it up, it will begin to pool at the lowest point of a container. This will become relevant later as we discuss designing a growing environment.
Fruiting substrate
Similar to bulk substrate, fruiting substrate is going to be very different depending on the type of mushroom you are growing, your budget, and the materials you have at hand. The goal with fruiting substrate is to provide a selective, nutritionally dense medium that will facilite high yield of fruiting bodies over one or more “breaks” or “flushes.”
While all the other parameters that we have discussed are still in play, this is generally the first stage in which the culture is exposed to normal, “unclean” air. The process of exposing the fruiting substrate to air is unique for each type of mushroom but generally follows the same type of process. I’ll discuss that in more detail under the “Fruiting Mushrooms” section.
Optimizing your substrates
You could honestly spend lifetimes trying to develop the idea substrate for a specific mushroom - I know I have. Each one will be different depending on the goals of the technician. Do you want higher yields? Lower contamination rates? Faster colonization time? One break? Multiple breaks? Lower cost? Vegan spawn? Each one changes how you design your medium. And while each substrate will be different, there are some common methods of production that carry through.
Before talking about substrates in detail, we should discuss how fungal mycelium interacts with its food. As mycelium grows, it acts like a giant inside-out stomach secreting various enzymes into its environment. These enzymes break down some of the materials it interacts with and the resulting compounds are then absorbed into the hyphal mass. For the most part, these enzymes remain in the substrate and continue to work until they are either broken down or locked up. Additionally, as they grow through the substrate, most fungi will decrease the pH. In general, free easy to access nutrition like simple sugars and amino acids certainly allow for quick growth but almost all other fungi and bacteria are going to be able to metabolize it faster than your culture of choice. Complex carbs, proteins, and fats are harder to break down resulting in slower growth but they are more nutrient dense. This poses some problems but they can be solved with some strategic planning.
With agar work, most people will use a premixed product like Potato Dextrose Agar (PDA) that is first completely sterilized. This is a fairly general medium containing easy to consume nutrients that will readily show both fungal and bacterial contaminants. This allows for a number of advantages. A standardized medium normalizes the range of morphological characteristics for a given strain and allows for contaminants to be readily identified and avoided.
Bulk substrate is where the culture should get prepared for its final fruiting substrate. Using a mix of easy access and complex nutrition, bulk substrate begins priming the mycelium enzyme battery to break down more complex substrates. Ideally, the bulk substrate should contain in some fraction, all of the ingredients that will be present in the final substrate. As the easy to digest nutrition is been used up, the mycelium begins secreting more enzymes to break down the more complex nutrition. During the next transfer into the fruiting substrate, these enzymes will carry over and begin working. This stage is generally takes the most time. Because of this, most bulk substrate is pH adjusted to prevent a premature pH crash midway through the growth cycle.
The fruiting substrate generally should be the most complex and selective medium possible. This helps reduce the prevalence of contamination once the substrate is exposed to air. Fortunately, if the bulk substrate closely matches the fruiting substrate, there shouldn’t be a significant lag in colonization and production due to the carried over enzymes. Additionally, the mycelium itself will continue to produce the correct enzymes as it colonizes the new substrate. As it grows, it also continues to acidity the medium. pH adjustment will also be required here.
In general for all substrates, there needs to be a balance between gut loading the mycelium,
Sterilization vs Pasteurization
All substrates that are not fruiting substrates should be sterilized, not pasteurized. Full stop. Any contaminants that survive pasteurization will inevitably be transferred into the next stage of the process. Therefore, it is imperative to ensure that the initial stages of the process remain as clean as possible.
Fruiting substrates are different and sterilization can be expensive. Some varieties like oyster mushrooms are incredibly aggressive and can compete very well against low levels of contamination. Varieties like these can tolerate pasteurized substrate very well and in many cases, do better than in sterilized substrates. Most mushrooms however, don’t compete as well and will still require sterilization. If you are going to experiment with pasteurization, this is the stage to do it.
While steam is the most common method of sterilization and pasteurization, pasteurization can be done with chemicals such as high concentrations hydrogen peroxide, lye or agricultural lime. These techniques are, in general, a bit more dangerous and complicated. There are some good guides if you want to give it a try but take all the necessary precautions. Also, just to warn you, if you are purchasing 35% hydrogen peroxide, you will be put on a list - it is nasty stuff.
Individual components
This is where things get tricky. Each mushroom has its own preferences. Each grower has access to certain materials at varying price points. There’s no one rule fits all. I’ll try to track down some of our more successful substrates for various mushrooms when I’m not in mobile.
Obtaining and processing your first cultures
Commercial options
Spores, agar plates, and grain spawn are all available commercially if you want to buy your cultures. For culinary mushrooms I recommend starting with grain spawn. It will generally be the best bang for your buck and can be used right away for bulk inoculation as well as culture work.
Agar plates are also good options as they are clean, cheap to ship, and can be bulked up without too much work.
Spores can be a bit of a pain. Spores will always have some level of contamination. Additionally, if you are unfamiliar with what a strain looks like on a particular substrate, identifying the differences contamination and your culture of choice can be difficult. Furthermore, unless you take the time to do a proper dilution in order to make single spore plates, you will have a wide variation of genetics present in your culture which can cause problems down the line (see Proper Development and Storage of your Cultures). That said, they are incredibly cheap to ship, easy to collect, imperative if attempting to breed, and the only way to obtain certain cultures depending on your local laws.
Liquid cultures
Liquid cultures are also another common option that offer a lot of advantages. They can come in the form of suspended mycelium as well as suspended spores. These are easy to use, cheap to acquire, and come in a wide variety of strains. That said, I would still transfer them to agar for the benefits listed above.
Self-obtained Cultures
While 9/10 times it is easier purchase cultures, you can isolate your own cultures fairly easily from other mushroom fruiting bodies.
PLEASE, please, please, do not collect mushrooms from the wild unless you can 100% identify them. You can make yourself extremely ill or worse if you select the wrong ones. Many mushrooms won’t kill you immediately, however the toxic compounds will build up in your body over time and can reach a deadly threshold. It’s not worth the risk.
Collecting spores
To collect mushroom spores, select the largest, prettiest mushroom from the lot. If it is not coming from the wild, select a mushroom from the middle of first break, without signs of blemishes. For cap varieties, there are several schools of thought. Some people believe that selecting a cap that hasn’t broken its veil, allows you to keep a cleaner culture. In my experience, that is not the case; instead I tend to have lower yield of spores. In general I select a mushroom that is mature, hasn’t flattened out, and appears to be laden with spores. Place the cap cut side down on a piece of tin foil in a sanitized still air box and let the spores drop naturally over the course of a few days.
From this point on you have options. You can package them for dry storage, mix them into water, broth, or buffer, or put them directly onto agar. My recommendation is to store the large majority, and then do one of two things: A) mix a small amount of spores into a small amount of water or neutral buffer. From there, using a hemocytometer, estimate the amount of spores present, and dilute the solution to a point where you can pipette or syringe 10-20 spores per agar plate. Once the germinate, immediately transfer them to their own fresh plate and mark them with a strain number. This is the first start of a proper breeding program. For most hobbyists it is impractical but could be interesting to those who are looking to get more serious. Or… B) mix them all into a small aliquot of sterile broth or grain and spawn them as you would any other culture. From here, plan on growing a few crops of a given mushroom looking for your ideal morphology. Once you have a few nice looking mushrooms from the middle of your first break, you can begin an easier isolation method. Which is…
Direct tissue culture
This process involves taking tissue from a pristine specimen mushroom and growing it on an agar plate. This has a number of advantages over spore work. When done correctly, your new culture will be an identical clone of the mushroom variety you selected. It’s behavior and characteristics should on average match those of the selected mushroom. It also functions as a pure line by containing only one set of genes unlike batches made with multiple spores.
Once you have selected a mushroom, stage a few Petri dishes as far up wind as possible within your laminar flow hood. Using a spray bottle of 70% ethanol spray down the surface of the mushroom being careful not to agitate any of the spores. Place the mushroom down wind of your petri plates. From there, gently tear the fruiting body in half to expose the tissue inside of the cap and stem. Using a sterilized blade or tweezers remove tissue from the stem of the mushroom. Take care to avoid any tissue closer than 2-5mm from spore producing structures or the outside of the mushroom. Place this tissue on the petri plate and observe for contamination.
Proper Development and Storage of Cultures
This next section of advice is likely going to be the most controversial, not because of the validity of the information, but because many will see it as unnecessary. While it is true that for many applications, these next steps would not be necessary, I am aiming to discuss best practices as they are applicable to the industry. If someone decides to turn their hobby into a business, having the ground work laid out at the beginning will serve them better in the long run. I am happy to discuss why I prefer one method over another, so please don’t hesitate to ask if you have heard advice contrary to what I have said.
Developing your culture.
Once you have your first culture, you need to start developing it. For me, that has several steps. The first is to make sure that the culture is clean from contamination. To do this, you can use selective media with and without the use of antibiotics or simply select areas of actively growing, identifiable mycelium and transfer those to new plates. The second step, is to make sure that your culture is monotypic. Over time in the hobbyist circles, mycelium and spore cultures become contaminated with spores from the same species as the original culture. These can go completely unnoticed and will affect the uniformity of your crops later on down the line. In order to ensure that you are isolating the pure intended mycelium, you must select the first 2-4mm of fresh growth from your cultures. Unfortunately, this can be difficult to do when the cultures are mixed, or if they are exhibiting strange morphological characteristics. That is why I recommend starting with a standardized medium such as PDA as they are more uniform.
Another complicating factor is the overall morphology of a culture. Until it has been worked for some time, culture morphology can be erratic. Each mycologist has a certain selection of characteristics that they are looking for in a culture and I would like to share mine.
Tomentose vs rhizomorphic growth
In general, I prefer to select for tomentose growth. Rhizomorphic growth is a staple for most hobbyist growers and those working with actives. It’s unique characteristics make it easy to identify and distinguish from many contaminants and I believe this has lead to its popularity. Unfortunately, in my experience, it has been associated with slower growth, erratic growth, mutation, and continued vegetative growth under fruiting conditions.
Tomentose growth is generally more even in its hyphae distribution. This makes it easier to detect aberrant sectors and select for more even margins. It also tends to colonize the medium in a more even spherical shape that may help with more efficient growth and substrate utilization. This also helps us when designing substrates, as any irregularities in growth characteristics can help guide us to their cause.
One example we had was from a shiitake culture that was extremely consistent. During a quality inspection, we noticed rhizomorphic growth forming on the upper edges of the bag. We went through our notes and couldn’t find anything wrong with the batch. We then started to see it on the next batch, and the one after that. After reviewing our cameras, we noticed that a delivery driver used some of our sawdust to soak up an oil stain he left on our blacktop; the before leaving, he swept it back into our piles. That made it into our spawn material and caused the issue we saw. Those bags still grew mushrooms, but their yields and quality were severely sub par.
This isn’t to say that rhizomorphic growth is bad. It is a useful characteristic that someone can select for to develop their culture beyond just using random agar wedges. That said, if given the opportunity to train new technicians, we try to teach selection for tomentose growth due to the advantages we tend to see. In the end, you need to have a vision for what your culture should look like and mercilessly select for it. It’s said that cultures take on the personality of their mycologist and it’s because of this selective pressure that each person applies.
To be continued…
