The mushroom "seed" (propagation material) is generally referred to asspawn. It is nothing but the mycelium of any species of mushroom which is in a vegetative stage of growth on a substrate media which can be anything like grain, saw dust, nutrient liquid, straw etc. The role of mushroom spawn are the further propagation of the vegetative phase of the mycelia so that a large cluster of substrate gets colonized with the mycelium and fruits mushroom when the conditions favor.
Availability of spawn culture
The availability of good quality spawn is the limiting factor for mushroom cultivation in many developing countries. Customs' bureaucracy, high shipping costs and the difficulty to keep the spawn cooled during transport, often hinders imports. It might therefore be necessary for the mushroom grower to produce his own spawn. If good quality spawn of the desired mushroom species can be obtained at a reasonable price, it would be wiser to concentrate on the mushroom growing process. If not, spawn will have to be produced or multiplied by the mushroom grower.
The complete procedure of spawn production involves preparation of the medium, filling the test tubes or petri dishes and sterilizing them, and the process of inoculating larger containers with this culture.
Spawn production requires a clean laboratory and specialized knowledge. Basically, spawn production is nothing more than putting mycelium of the desired mushroom in suitable sterilized substrates under aseptic conditions.
In practice, however, producing spawn is not that simple. Suitable strains from the required mushroom species have to be maintained under strict conditions to avoid degeneration. If this is not possible, tissue culture from a fresh and healthy mushroom should be used for spawn production. In addition, the spawn production room has to be kept meticulously clean to avoid any contamination.
The starter culture
The starter culture (or mother culture) can be made from a fresh and healthy fruiting body or obtained from a spawn producer or laboratory. More agar cultures are then made from this starter culture. These serve to inoculate larger containers (like bottles) with mother spawn, which can be used to inoculate the final spawn substrate.
The minimal requirements for a spawn production unit are:
a) A sterilization unit (pressure cooker, autoclave)
b) Sterile environment: inoculation box or laminar airflow cabin c) laboratory equipment like petri dishes, test tubes, scales, alcohol, flame
d) Incubation room
The above equipment is commonly available in hospitals, research stations and universities.
The raw materials include:
i) Ingredients for media preparation
ii) Substrate material (grain, wooden sticks (skewers), sawdust, or even oil palm fruit fibre
iii) Pure culture or fresh mushroom of the desired mushroom species strain iv) spawn containers (such as bottles or plastic bags)
In areas lacking mushroom production, spawn may be obtained from a spawn producer, a university or a research station at the start of a project.
The sterilization process
Grain, sawdust and compost contain large n umbers of contaminants. A single grain kernel may contain thousands of bacteria, fungi and actinomycetes. Every one of these undesired agents, which are called contaminants, is capable of spoiling substrates that have not been properly sterilized or inoculated under unhygienic conditions.
A heat treatment of 15 minutes at 121 °C is usually sufficient to kill all organisms. It takes quite some time for the steam to heat the inner core of substrates to this temperature, depending on the way the sterilization/ pasteurization unit is filled and on the capacity of the burner. Steaming in an oil drum for at least 6 hours is usually necessary to ensure proper heating of the inner core of the substrate bags.
The cheapest option is to obtain one or more large pressure cookers.
Select pressure cookers that maintain the pressure when the final temperature has been reached. The simplest pressure cookers blow out steam when the pressure is too high. The pressure inside will then often drop below 1 atmosphere overpressure, causing the media to boil. This should be avoided. Petri dishes or bottles with agar media may become messy if this type of pressure cooker is used. The pressure cookers should have an inside rack, which will effectively ensure a more even temperature distribution inside the pressure cooker. The heat source is either external (gas burners, coal, wood) or built-in (electric).
Tips for Safe Operation of Pressure Cookers
Read the instructions. Improper operation is a real threat to your safety, so it’s imperative that you know everything possible about your unit. Every unit operates differently. Even the same brand changes from year to year, so stay apprised of all the features of the model you have purchased.
Make sure the cooker has enough liquid.
Running the cooker dry can ruin the contents and potentially damage the cooker. To make sure it always has enough water, I stack ring bands underneath the bottom standoff plate. This increases the volume of water in the cooker without bringing the water level too near the tops of the jars. For larger cookers, I often stack two levels of ring bands under the standoff plate if additional water is needed. You can use other items besides ring bands, but the goal is to raise the standoff plate an inch or two while the unit is in operation.
Make sure your juggler is not stuck to the vent pipe.
Most manufacturers recommend that you apply your juggler weight to the steam vent only after steam has been coming through it fairly steadily for several minutes. If you begin to cook with the weight on, it will sometimes stick to the lid and not become unstuck until the pressure gets to 20 psi or higher. If this happens, the juggler could blow off the unit entirely once it breaks free.
Release the pressure safely. Let the pressure decrease from the cooker naturally. I used to pour cold water on the hot cooker or soak it in water to speed up the cooling process, but this isn’t the safest method. It’s recommended for some models, but others expressly recommend against it.
Never move or open the cooker until the pressure is all the way down. If the pressure has not diminished, a large burst of steam or a small explosion may occur. Steam causes serious burns. Be patient.
Always stay near the cooker while it’s in operation. Consistently monitoring the cooker is one of the best ways to ensure safe operation. Most of my close calls with the cooker have occurred when I was distracted while the cooker was in operation. The cooker’s pressure built unimpeded until it popped the release valve and let steam escape. If this goes on for too long, the cooker will run dry, ruining the contents and potentially ruining the cooker.
Keep kids and animals away from the cooker while it’s in operation. This one is self-explanatory. Maintain the cooker. Follow all manufacturer instructions for maintenance. For Presto and Mirro cookers, this means making sure the gaskets don’t need to be replaced. For All American cookers, apply a small amount of petroleum jelly to the metal to-metal seal. Be sure you know the needs of your individual cooker.
Never put jars or bags directly on the bottom of the cooker. Always use the standoff plate that came with the cooker. The contents will burn without the standoff plate.
Clean Environment
A clean environment is absolutely essential to spawn production. In particular, whenever the containers with sterilized media need to be opened it must be done under aseptic conditions. The air carries numerous contaminants, which easily infect the sterilised media. It is therefore necessary to use special cabinets and inoculation rooms for performing the handling and the preparation of the tissue) cultures.
Inoculation rooms
The interior of the inoculation room should consist of nonbiodegradable materials. All the surfaces should be smooth and easy to clean. Shelves should be designed in such a way that the floor beneath can be cleaned easily. Shelves are typically made of galvanized iron or Formica. Inoculation cabinets
These simple inoculation cabinets are widely used all over the world. They can be constructed cheaply from locally available materials. The front glass pane can be opened to fill the cabinet with the sterilized media. The inside is disinfected by cleaning with a 2% Formalin solution or 70% ethyl alcohol. Take care when using these chemicals. Some of them are poisonous and/or irritating to nose and eyes. Cautiously follow the instructions to ensure safe use.
Laminar airflow cabinets
A laminar airflow system (LAF) consists of a fan, a duct, a HEPA (High Efficiency Particle Air) filter and a hood. In laminar airflow contaminants can spread in only one direction. In turbulent airflow it is possible that spores move in different directions, causing more contamination. The ventilators are rated by the producers according to the volume of air they can blow through materials of specified resistance. About 0.45 m/s air velocity is considered best for good laminar airflow.
The fan should be regulated stepwise and have the capacity to push double the amount of required air through the filter to reach the required air velocity, to account for pressure losses when the filter gets loaded with particles. The filters and ventilators are the heart of any laminar airflow system, but other factors have to be considered too: the operating persons, their skills and their hygiene; the construction of the ducts and filters to ensure that no contaminated air can be sucked in.
The first steps in spawn production are performed on artificial media. These should contain sufficient nutrients for the mushrooms to grow, like saccharides and a solidifying agent (agar or gelatin). The mycelium grows on the surface of the medium and will later be used to inoculate larger amounts of substrates like sawdust or grain. Test tubes or petri dishes (or flat milk bottles) can be used as culture containers. Instead of working with cultures, one could also try to purchase small amounts of good quality mother spawn to prepare the final spawn.
Flow Hoods
Sterile techniques in mushroom cultivation, such as making syringes, simple agar work, grain-to-grain transfers, and other simple procedures. I used a glovebox for my first 3 to 6 months of mushroom cultivation.
But if you plan on sticking with the hobby for a while and want to try some of the more advanced methods, you’ll need a laminar flow hood.
A flow hood is essentially a large box that sits on a table with a high-efficiency particulate air (HEPA) filter facing you, and a fan forcing air down through the filter. This setup creates an area of sterile air directly in front of the filter, and allows you to perform sterile procedures with unrestricted movement in an open-air situation. The goal of these filters is to create a laminar flow, a series of air currents emitted straight out of the filter across the work surface, without mixing with other side currents.
Although they can be quite expensive a flow hood pays for itself many times over in terms of time saved and contamination averted.
I cannot emphasize enough how much easier mushroom cultivation is if you have a flow hood at this point in your education. You can probably find your way through most of the processes in this intermediate section without a hood, but I’m sure you’ll start to see the benefits of one as you begin to work through these processes without it. If you have some construction skills, it’s fairly easy to build one yourself.
Tissue cultures
Young and vigorous mycelium can be obtained from a young fruiting body using a scalpel, alcohol, sterilized agar slants, petri clishes or bottles with agar, flame (non-smoking), and a clean table to work on, or preferably a laminar airflow cabinet or inoculation box.
i) Wash the mushroom thoroughly.
ii) Dip the scalpel in alcohol, and then flame it until red-hot.
iii) Let it cool down for 10 seconds.
iv) Now break or tear the mushroom lengthwise (do not cut it with a knife, since contaminants from the surface can stick to the blade). Do not touch the inner surface with your hands.
v) Use the heated scalpel to remove a small piece (2x2 mm2 is sufficient) of the inner tissue. Take care that no outside surface tissue is included.
vi) Open the test tube/petri dish.
vii) (When using test tubes: heat the mouth of the tube in the flame to kill unwanted spores). Then, gently replace the tissue on the scalpel in the middle of the agar.
viii) Immediately replace the plug.
ix) Inoculate at least three cultures, but preferably more. Incubate the newly inoculated agar slants or petri dishes at 25 °C for about ten days. Within three to four days mycelium will cover the tissue and branch out on the agar. If no growth occurs on the agar, check the following:
a) Possibly the mushroom was too old. Try a fresher specimen.
b) Possibly the scalpel did not cool down before taking the tissue sample, thereby overheating the mycelium. The mycelium should be white and grow out from the tissue. If yellow, blue, green or grey mycelia form on other places on the surface, then these are fungal contaminants. A creamy, shiny growth often indicates bacterial contamination.
Preparation of media
Most species grow on the following media:
- Wash and weigh the potatoes and cut them into small pieces.
- Boil for about 15 to 20 minutes until they are soft.
- Remove the potatoes.
- Add water to the broth to make exactly 1 liter.
Potato Dextrose Agar (PDA) medium
Ingredients: 200g sliced potato, 20 g agar powder, 20g dextrose or ordinary white sugar, 1 liter water.
- Add the dextrose and the agar. Be sure to add the right amount of sugar and agar, otherwise the medium will become either too soft or too hard.
- Stir occasionally and heat gently until the agar has melted. The agar should be hot when poured into the test tubes or bottles otherwise it will become lumpy.
- Fill about one fourth of the test tubes.
- Then, seal the tubes or bottles with cotton plugs.
Rice bran broth medium
The above recipe for PDA is commonly used for culture preservation, but for multiplying cultures, the following recipe is cheaper and easier to prepare. Ingredients: 200 g rice bran, 1 liter water, 20g gelatin. Boil the rice bran for about 10 minutes in the water. Filter, save the broth and melt the gelatin and pour into bottles and sterilize.
Preparation of slants
After filling the test tubes or bottles with the medium, they must be sterilized before they can be used. The most commonly used sterilization units in small-scale laboratories are pressure cookers, but autoclaves can be used as well.
Procedure
i) Pour water into the pressure cooker to the level of the rack.
ii) Place the bottles/test tubes in the racks with a plastic covering to prevent water from wetting the cotton plugs.
iii) Then close the lid firmly.
iv) The air vent should be open at the beginning to allow the air to escape. This will take some minutes from the moment of boiling and steam escape.
v) Close the air vent. A pressure gauge shows the pressure rise.
vi) Sterilize under pressure for 20-30 minutes.
To increase the surface area, the test tubes or bottles are placed in an inclined position when the agar is still fluid. Take care that the agar does not touch the cotton plug otherwise it might become contaminated. Do not move or handle the test tubes until the agar has solidified, otherwise a small portion of the agar may solidify at the other side of the slant or too close to the plug.
Sub-culturing
Inoculate more test tubes using the methods discussed above. For reasons of degeneration it is advisable not to transfer from one mother culture more than eight times or to use mother cultures on agar for more than two years. The mycelium will degenerate after a certain number of transfers, so it is not possible to keep on transferring the cultures on agar forever.
Spawn containers
Spawn containers should be made out of heat resistant material: mostly glass and polypropylene (PP). The spawn containers have to be tested to see if they can withstand the temperature inside the sterilization unit. If the pressure is greater than 1 atmosphere overpressure, the temperature will be higher than 121° C. PP bags sometimes crack easily after having been subjected to the sterilization process. Avoid bags with seams: these tend to split after the heat treatment. Glass or heat-resistant plastic bottles are often used for the mother spawn.
Wide-mouthed jars, milk bottles and dextrose bottles can also be used. Dextrose bottles are ideal, because they can be obtained for free from hospitals and they have air outlets that can easily be plugged with cotton wool. They can be used for final spawn too, but if the mycelium from the spawn material has grown into one big clump, the bottles have to be broken to get it out.
Polypropylene bags with cotton plugs (or filters) to allow aeration are much in use for the final spawn (both sawdust and grain). Their size varies between 2.5 and 15 litres for grain spawn. The exchange of metabolic gases like co, with ambient air has to be ensured; unwanted spores, however, must be prevented from entering the container.
Mother spawn
Mother spawn can be used to inoculate either grain spawn or a second generation of mother spawn. In simple laboratories, grain mother spawn should not be used to inoculate another generation of grain mother spawn because the risk of contamination and degeneration will be too high.
Preparation of grain spawn
The main advantage of grain is that it is very nutritious for fungi and forms kernels easily. The kernels can easily be dispersed in the substrate. The main disadvantage is that it provides an optimal substrate for other organisms too. The chances of contamination are therefore much higher compared to sawdust spawn.
A final consideration for which grain to use is the surface area of each grain. Grain surface area affects the speed of colonization. Small grains, such as millet, fit closely together in the jar; consequently, they have a very large total surface area to colonize. Larger grains, like popcorn, have a smaller total surface area, with many less units in the same volume. If all other factors are equal, the larger surface area will need more time to achieve 100 percent colonization.
The differences in colonization times are not staggering, but they can be important in some circumstances. For example, a jar of popcorn will probably reach 100 percent colonization several days faster than a jar of millet. As with most things in life, there’s a tradeoff for faster initial colonization time.
Usually, colonized grains in quart jars are broken up and spawned (transferred) to another substrate. Small-kernel grains, such as millet, will break up into many more individual pieces than larger grains, like popcorn, if you start with the same volume of each. These smaller grains offer some advantages for certain methods: more individual colonized kernels, for example, mean more individual inoculation points in the substrate you’re transferring to, and ultimately faster colonization of this transfer substrate. The mycelia will not need to travel as far to meet the nearest startup colony. For many of the methods discussed later in this book, such as using bulk substrates, more but smaller transfer colonies will yield faster colonization
Thwarting Endospores
Soaking your grain for 12 to 24 hours ensures adequate hydration, but it also has another benefit: it allows bacterial endospores to germinate, and thus become more susceptible to sterilization temperatures. Pressure cooking grain is necessary because bacterial endospores can survive temperatures of 212°F (100°C). Sterilization at 250°F (121°C) for 20 minutes is required to kill these endospores.
Remember that the entire contents of the jar must reach that temperature for the recommended time. The interior of the jars or bags takes longer to heat up than the outer areas, so actual cooking times must be longer than 20 minutes to ensure proper sterilization of the entire substrate. If you’re having problems with bacteria in your grain jars, you may want to try longer sterilization times and/or switching to the soaking hydration method.
Kinds of grains :
Different grains can be used such as wheat, rye, millet, rice or sorghum.
Boil grain first, drain, then fill in containers and sterilise. The moisture content of the grain, after boiling, should be around 50%. If it is higher, mycelial growth may be faster, but the danger of wet spot bacteria is also greater. If it is drier than 35% mycelial growth will be rather slow.
Grain spawn formula
Grain in small containers can be moistened to a higher degree than grain in 15 litre bags. For 2 liter containers, use the following recipe: 480 g rye, sorghum or wheat, 400 ml water, 2 g gypsum (45% moisture).
Preparation of sawdust spawn
3 Sawdust spawn substrate: Sawdust 10 kg, CaCO,147.5g, Rice bran 1.25, Gypsum 0.1475g, Urea 0.5 g, Water 1.5 liters.
Sterilization
Sterilize the spawn containers in an autoclave. The length of time depends on the autoclave, the way the spawn containers are packed(dense or loose) and the size of the containers.
For instance, two hours for 500 g containers; three to four hours for 3 kg bags. Shake the bottles when taking them out of the autoclave or the pressure cooker.
Preventing Common Problems
When pressure cooking or boiling any of these grains, be sure to avoid overheating, which can cause grain kernels to burst or explode. When kernels explode, they release starches that can cause your grains to clump and can also make bacterial contamination more likely
Sometimes rye grain can become stuck together and clumpy after it has been pressure cooked. To prevent this, remove the jars from the cooker during the cool-down process, while the cooker is still warm but the jars are cool enough to handle, and shake them to mix up all the kernels in the jar. Shaking the kernels at this time will keep them from sticking together. Remember that jars suck in air as they cool, so let them finish cooling in front of a flow hood.
Inoculation
Once the temperature in the center of the container has dropped to below the maximum mycelial growth temperature, the spawn containers can be inoculated. Use at least one (for 250 ml bottles) or two (for bigger bottles) squares of 10 x 10 mm2 from the full-grown agar of the mother culture for each bottle.
Incubation
Incubate the bottles until the mycelium has grown all over the substrate. The temperature should be close to the optimal temperature for mycelial growth. Shake once (after eight days) or twice during the incubation period (or every three or four days) to distribute the mycelium evenly and to prevent kernels from sticking together.
It will usually take 8 to 12 hours for your cooker to cool completely. Many sources of inoculum can be used, including spore syringes, liquid culture syringes, agar, and grain-to grain transfers. Shake your jars after they have been inoculated to distribute the inoculum throughout the jar.
Shaking Jars
There are a couple of reasons to shake jars during the grain spawn process: the first is to prevent the grains from clumping in the jars; the second is to speed up the colonization process. Grain is most likely to clump in a jar and needs to be shaken just after it’s pressure cooked for the first time. This is especially true if you’re using smaller grains, such as rye and birdseed; it’s less likely if you’re using popcorn. The smaller grains may become hard to break apart if you don’t shake them while they’re still warm from the pressure cooker. If they’re not broken up then, the inoculum will have a harder time evenly colonizing the jar.
There are many ways to break up the contents of a jar. Sometimes simply shaking the jar forcefully up and down is sufficient to break up the interior contents. (Be careful not to displace the filter in your lid during this process!) Shaking by hand isn’t sufficient for many grains, however, especially if they’re fully colonized. Instead, you’ll need to find an object that you can use to make contact with the jar. I find that a tennis shoe or a spare tire works best.
Jar Colonization
After your jars are inoculated, it usually takes a week or two for them to become fully colonized. The amount of time it takes depends on the process you used to inoculate and the mushroom species you chose. During this colonization time, you need to find a warm, dry, dark place for your jars to sit. The ideal temperature depends on the species, but most species do best at 70 to 75°F (21 to 24°C).
Colonizing jars can sit at room temperature on a lab shelf and do just fine. You could build an incubator to warm them up to the ideal temperature (see chapter 4), or find a cool place if the ideal temperature is lower than room temperature, but the truth is that, for most species, “ideal” temperatures will speed up colonization by a day or two at most. This might be important if you’re on a strict schedule, but for most home growers, it isn’t an issue.
While you wait for the jars to colonize, the key thing to keep an eye out for is the appearance of colors other than the mycelium you have injected. Most mycelium is white, although some common edible species can be black, gray, or even orange. Green is universally bad, because it’s usually a mold called Trichoderma or a species of Penicillium.
If your grains appear to be wet in the jar, it may mean that a bacteria called Bacillus has colonized your grain. This is probably the most common problem I’ve encountered, more often even than mold contamination .
If bacteria have colonized your jar, their growth will inhibit the growth of the mycelium you’re trying to propagate. After it has been inoculated, there’s little you can do to save a jar that has been infected with bacteria; you’ll probably just have to throw it away. One simple way to test for the presence of bacteria in the jar is to smell the filter. If it has been colonized with bacteria, you may detect a sweet or foul smell.
Storage
Keep the spawn in the refrigerator (except for certain strains of Pleurotus djamor, Volvariella volvacea, spawn, which are cold sensitive and should be stored above 12 °C) and only take it out when needed. Grain spawn can spoil in one night at temperatures above 25 °C.
Another Way
Another way most farmers opts for is they directly purchase first generation spawn from us by visiting our online storewww.bmmushroom.shop
At our lab we maintain the vigorous high yielding strains of verities of mushroom. We provide Pan India and worldwide shipping options.
One more thing, mushroom cultivation is not all about spawn, it is just a part of the cultivation. The biggest factor upon which the success of mushroom cultivation depends upon is knowledge. You can learn about cultivation by joining the flagship mushroom cultivation course.
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