Liquid Culture Guide: Everything You Need to Know About Mushroom Cultivation

Liquid culture (LC) provides a means of supercharging your fungi cultivation efforts. It consists of fungal mycelium suspended in a liquid medium, which can promote faster colonization than starting with spores or colonized agar. This simple hack is the most efficient way to expand fungal biomass for mushroom cultivation. It will save you money while unlocking new levels of possibility in your mushroom-growing journey. It is cost-effective and extremely scalable, with a little LC going a long way. 

Liquid culture syringes make it easy for you to get started in mushroom cultivation, allowing you to skip the most delicate part of the process so you can focus on growing. Another important benefit of LC is that it allows for easy stress-free inoculations that can be performed in the open air, side-stepping the need for more specialized equipment, making it easier than agar work. Creating LC is a different matter, but the procedure on how to do this is outlined in the following guide.

What is Liquid Culture?

Liquid culture (LC) comprises sugary water that has been sterilized and then inoculated with mycelium or spores to produce mycelium suspended in a liquid growth medium or broth. This can then be used for inoculations onto a substrate such as grain. It tends to comprise water with a 4% sugar content, and sugar sources may include malt extract, corn syrup, dextrose or honey (a higher sugar content than this may impede mycelial growth). 

While using liquid culture to perform inoculations does not require an aseptic environment (with correct hygienic inoculation procedure in place), creating LC does necessitate this, such as using a glove-box or laminar flow-hood. However, once you have a confirmed clean LC culture, it can make for easy stress-free inoculations in non-sterile environments in the open air without risking contamination. Liquid culture jars require special lids that contain a fine micron filter patch and a self-healing injection port that allows the growing mycelium to breathe while keeping contaminants out, while also allowing for easy transfer of the LC.

Benefits of Liquid Culture

Liquid culture offers more predictable outcomes and faster colonization than inoculating with spores or agar culture. Inoculations with LC are simpler than those with agar, which is a more intricate process that can be overwhelming for novices.

Given that liquid culture is made up of growing mycelium, it can cut down on growing time in comparison to starting from scratch if using spores, which can take time to germinate. Using LC allows one to hit the ground running when growing fungi, helping reduce incubation time. 

Learning to create your own liquid culture means you will not need to purchase or create nearly as many spore syringes or agar plates for inoculations. It is cost-effective, having low production costs due to its basic ingredients, and it is extremely scalable (being particularly well suited to larger scale cultivation).

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Expanding LC is very simple, and the creation of LC can simplify the beginning stages of mushroom cultivation. Liquid culture taps into the inherent and unlimited capacity of mycelium to grow and expand, making it a convenient and efficient way to propagate mycelium for fungi cultivation. A little LC also goes a long way, with 1.5 milliliters of LC being more than sufficient to inoculate a grain jar, so a single 300 ml jar of LC could potentially inoculate up to a minimum of 200-grain jars. Once mastered, liquid culture techniques can also be successfully applied to cultivate many different types of fungi.

Another important benefit of using LC is that grain inoculations can be performed on an open-air surface such as a kitchen counter, eliminating the need for specialized equipment such as a still air box (SAB), glove box, or laminar flow hood (creating LC is a different story, however). This means that once a clean culture of LC has been prepared, contamination risks are very low if following the correct aseptic procedure when performing inoculations.

Drawbacks of Liquid Culture

While inoculations with liquid culture can be performed in an open-air environment, creating one’s own liquid culture will require access to an aseptic environment provided by an SAB, glove box, or laminar flow hood due to its sensitivity to contamination.  If new to this, some trial and error will likely be required before you can confidently produce clean LC — but these initial efforts will bear fruit for the patient and persistent grower. Purchasing a syringe of LC and then expanding it yourself sidesteps this challenge if you are new to LC.

One drawback of liquid culture is that in marked contrast to agar, which shows up as well as contaminated (which can then be dealt with), it is hard to tell if it is contaminated, as contamination can hide in the suspension. However, agar can be used to test LC for contamination. 

What is the difference between liquid culture and a spore syringe?

A syringe of liquid culture harbors live mycelium that is growing and raring to go, which allows cultivation projects to hit the ground running. By contrast, if using a spore syringe, the spores must first germinate before the mycelium begins growing, which adds further time to grow projects. While a single spore syringe may only be sufficient to inculcate 5-6 grain jars, a single jar of liquid culture can inoculate hundreds of grain jars. However, a spore syringe can be used to inoculate liquid culture.

What is the difference between liquid culture and agar?

Agar is a gelatinous substance derived from seaweed and is a commonly used medium for germinating spores and expanding and storing mycelium cultures. Inoculation using agar requires at minimum a semi-sterile environment like a good SAB, or a glove box or laminar flow hood. Agar also provides a solid surface to provide better observations of mycelial growth which can be an ally in strain selection work. Agar also makes it much easier to spot contaminants than liquid culture, but inoculating agar will spores, and inoculating grain jars with colonized agar necessitates aseptic conditions and specialized equipment, which inoculations via LC do not require.

Supplies You’ll Need to Get Started

To start producing your own liquid culture, you will first need to source some supplies. These include: 

A pressure cooker capable of cooking at 15 PSI. Jars (500 ml Kilner or Ball preserving jars work well, but old pickle jars can also be repurposed). 10 / 20ml luer-lock syringes 18 gauge luer-lock syringe needles Clear RTV silicone Synthetic filter discs Light malt extract powder Karo corn syrup A torch lighter (ideally, other lighters will work) 70% isopropyl alcohol A scale (sensitive to 0.01g) Some shards of glass, marbles or ball bearings

To modify the jar lids, you will need access to a hammer and a screwdriver or a drill with a sharp 1/4-inch metal drill bit. If you want to create your own LC, you will need a good still air box (SAB) or glove box. You will also need agar to create your own LC from spores. All of these items can be sourced online. You will require larger quart jars for grain inoculations.

Creating liquid culture jars for inoculations

In order to use liquid culture, it is necessary to modify the lids of the jars that will contain the LC and the grain you will inoculate with it. These liquid culture jars require special filter lids that contain a self-healing injection port in addition to a fine micron filter patch that allows the growing mycelium to breathe while keeping contaminants out, and allow for easy transfer of the LC to be performed repeatedly in a manner that will resist contamination in open air.

Creating Filter-Lids

A flow hood or glove box is unnecessary when a sterile liquid culture syringe skillfully employs filter lids fitted with self-healing injection ports. Creating these lids is simple, with tools and supplies readily obtained online or from a typical hardware store, and dozens of durable, reusable lids may be created in a single  session. The lids are fastened on regular-mouthed or wide-mouthed glass canning  jars, which serve as the final incubation vessels.

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Creating filter lids with self-healing silicone injection ports is essential for producing liquid culture, expanding it, and performing subsequent inoculations of grain jars. Some cultivators have successfully used 13 mm (0.22 um pore) syringe filters for gas exchange in liquid culture jars, as they allow gas but not liquid to pass through the filter membrane. This allows the jars to be shaken to distribute the mycelium and prevent it from forming clumps that will block a syringe needle. However, some cultivators (myself included) have experienced vacuum-lock issues when trying to uptake mycelium from jars with syringe filters. Swapping the syringe filter for a small disc of fine micron synthetic filter discs (which are sold online for this purpose) can eliminate these pressure-lock issues. For grain jars, compressed/stiffened felt (“EZ-Felt”) or Tyvek may also be used as grain-spawn jar lid filter material.

Liquid Culture Lid Tutorial:

Select a new metal canning jar lid. The first task is to create two holes in the lid. There are three options here:

With the lid screwed into place on the jar, using a hammer and a screwdriver (placed vertically over where you want the hole), make two holes in the lid. Use a small punch and die set (which goes up to a 1/4 inch) to punch two holes in your jar lid. Place the lid on a wood block and drill two holes using a high-speed drill and a sharp 1/4 inch metal drill bit. You may make a small indent with a punch to direct the drill bit first. Be very careful and start slowly, as the drill bit may get stuck, causing the lid to spin off and cut your hands. Use a rosebud or a sharp utility knife to remove any burrs. If using plastic rather than metal lids with your jars, apply gentle pressure with the drill, or else you risk cracking the lids.

Use clear high-temperature RTV gasket-maker silicone to create the injection port and fix the filter patch (clear silicone used to make the injection port will make performing inoculations easier). When using silicone, it’s wise to put some kitchen towel down, as it is sticky! Wipe the lid with 70% isopropyl alcohol to ensure good adhesion of the silicone. Once the alcohol has evaporated, place a blob of RTV silicone over one of the holes. To create the injection port, turn the lid over and repeat the same process on the other side, producing two blobs that meet each other through the hole.

For the filter disc, on the filter disc material, trace a pen around something small and circular, such as a coin, and cut it out. Apply silicone to the outer circumference of this disc, and then firmly tamp it down over the other hole in the lid. Then repeat the step of applying silicone to the outer circumference of the filter disc on the surface of the lid, so that the silicone covers all of the outer edge of the disc. Turn the lid over, and ensure any excess silicone is not blocking the underside of the disc (this can be removed by something fine like a scalpel blade or metal hair clip). Allow the lids to cure for 24 hours – it is then ready for sterilization.

Creating liquid culture (recipe and procedure)

LC Recipe

So, having prepared your LC jar lids, we can now move on to preparing the LC itself. If you have received a syringe of liquid culture to get started, the first talk will be to expand this. This is the recipe for a 500 ml jar (300 ml of liquid per 500 ml pint jar is a good amount) of liquid culture but can be easily scaled up depending on needs. Don’t fill jars more than two-thirds full to allow for swirling the LC while avoiding getting the synthetic filter disc material wet (some may be water-repellant, so this may not be an issue). 300 ml of liquid per 500 ml pint jar is a good amount. Put a few shards of glass, ball bearings, or marble in the jar before sealing and sterilizing to agitate and break up the mycelium before extracting with a syringe (this will also aid in colonization by helping to break up agar wedges when inoculating LC solution). While some cultivators used magnetic stirrers for this purpose, using these items offers a viable low-cost alternative.

While there are a variety of LC recipes, this one is reliable. Light malt extract is favored for its nutrient profile and clarity after sterilization. If possible, try to use filtered or distilled water when making up LC nutrient solution. Some growers do report using tap water without issue, but mycelium isn’t much of a fan of the chlorine found in it. All of the required ingredients can be sourced at the grocery store or online. Heat and stir the following in a pan on the hob until the ingredients have dissolved completely:

– 300 ml water

– 6 ml of light Karo corn syrup

– 0.6 g Extra Light Dry Malt Extract

If making up larger batches, 1g of light malt extract (or 6 teaspoons / approximately 24 ml of honey) can be added to 600ml of water. An empty syringe can help measure out the corn syrup, and a scale will be required for measuring out the malt extract. Malt extract should be stored in a well-sealed jar with a desiccant pack, or else it can take on moisture and solidify, making it hard to use.

All nutrient substrates (liquid culture, grain spawn, agar, etc) must be sterilized before inoculation to prevent the growth of competitor bacteria and fungi. Pressure sterilization destroys fungal spores and bacterial endospores through prolonged steam heating at high pressure using a pressure cooker. A sustained temperature of 121C is possible at 15 PSI, quickly destroying all potential contaminants. Pressure sterilization is quick and convenient, permitting same-day inoculation of substrates and ensuring full sterilization when the correct procedure is followed.

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Sterilize the LC jars in a pressure cooker (for 30 minutes at 15 PSI  once it reaches pressure). Do not overtighten the jar lids to allow for contraction on cooling. Do not exceed the pressure cooking time, as over-cooking the sugar will lead to caramelization of the sugars, which will be detrimental to mycelium growth (the LC will take on a more amber hue as a result of caramelization — ideally, it should be the same color coming out of the pressure cooker as going in)

Wait for the jars to cool completely before inoculating, or simply leave the pressure cooker to cool overnight. Once the jars have cooled, apply sellotape around the circumference of the jar lid, between the lid and jar (as an added precaution, permitting entry of bugs). A 300 ml jar of liquid culture can inoculate up to at least 150 grain jars. Once fully colonized at room temperature, store LC jars in the refrigerator, where they may remain viable for up to 2 years.

Creating LC from scratch – Inoculating Liquid Culture with Agar 

Creating LC through inoculation with agar is more sensitive to contamination than performing inoculations with ready-made LC, so extra attention should be paid to the aseptic procedure. This includes disinfecting your work space, hands and arms, and all tools, agar vessels, and liquid culture jar with isopropyl alcohol. Wearing a surgical mask is a good idea. This should be conducted in a glove box or a good still air box (as a bare minimum), or in front of a laminar flow hood.

Having made up some agar using the information above and following its inoculation with spores, wait until you observe a healthy growing mycelial culture on the agar that is free of contamination. Flame-sterilize your scalpel or inoculation tool until the tip glows red. There is no need to wait for it to cool, as it will cool immediately upon contact with the agar. Select a clean, pure white region of mycelium from the outer leading edge. You may make several cuts and transfer a few wedges to increase the speed of colonization. Working very quickly, open your liquid culture lid and tap the scalpel on the rim of the jar to knock the agar wedge into the liquid. Close the lid immediately. Label with the species/date (permanent marker works well for this, and can be removed with IPA) and incubate at room temperature, or between 70-80F.

How to use LC – Performing inoculations

Once your modified jars contain sterilized nutrient solution, you can perform inoculations. A simple thing to remember is that any syringe and syringe needles making contact with the inside of your jars via their injection ports to uptake or inject LC MUST be sterile. This can be achieved using a combination of pressure cooking syringes and needles, swabbing (with 70% isopropyl alcohol), and flaming any syringe needles immediately prior to performing an inoculation. The benefit of using self-healing injection port lids is that they allow for sterile inoculations of both liquid culture and grain spawn without using a flow hood or glove box. Injection ports are simply wiped with 70% IPA and injected with a sterile liquid culture syringe needle. 10 – 20 ml luer-lock syringes with 18 gauge needles work well, and they can be sterilized and reused repeatedly. These needles are wide enough to allow uptake and injection of LC, but not large enough to damage the injection port with repeated use.

When performing inoculations, maintaining an aseptic technique is vital to minimize the chances of contamination. It is best to work indoors and close windows and doors to minimize airflow and airborne contaminants. Hands and arms should be washed thoroughly, and clothes should be clean. Wearing a surgical mask is a good idea. It is good practice to disinfect your work area by wiping surfaces with 70% IPA. Some advocate use of disposable nitrile gloves, but this may not be necessary if using 70% IPA to disinfect hands and arms (this process can be repeated as and when necessary).

Used Luer-lock syringes and needles can be sterilized and reused repeatedly. Place the syringes (with needles attached) in a wide-mouthed quart-sized canning jar with about a tablespoon of water at the bottom (to prevent dry heat). Cover with a grain-spawn lid and tin foil, and sterilize in a pressure cooker/canner for 20 minutes at 15 PSI. The syringes will be clean and ready to extract liquid culture whenever you need them, but they can also be used almost fresh from the pressure cooker as the plastic they are made from does not retain heat.

Inoculating Liquid Culture with a Spore Syringe 

A spore syringe can be used to inoculate a liquid culture. Shaking the spore syringe well to ensure the spores are well distributed, inject 1ml of the spore solution into the liquid culture jar via the injection port while following the aseptic inoculation procedure. Ensure the syringe needle is swabbed, and flamed, and the injection port is swabbed before and after injecting the spores.

Inoculating Liquid Culture with a Liquid Culture Syringe 

To preserve a liquid culture for future use, it is recommended that a new liquid culture jar be created in order to expand from a liquid culture syringe. This will permit dozens of future inoculations, and the creation of new liquid culture syringes. When your jar is nearing empty, simply create a new one and transfer a few drops of the old liquid culture into the newly prepared jar.

If you have purchased a syringe of LC, you can (hopefully) assume that the interior content will be contaminant-free (so only the needle will need sterilizing). If inoculating jars with your LC, you will need to sterilize a syringe before usage in your pressure cooker, following the steps outlined above. Disinfect the entire surface of your master LC jar and your freshly sterilized LC jar, taking extra care to wipe the injection ports. 

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When performing an inoculation, wipe the syringe and needle with alcohol. Wait for the alcohol to evaporate completely,  then flame the needle tip until it glows red. Wait 10-15 seconds for the needle to cool, or dip the tip in a small dish of alcohol. Wipe the injection port on your master LC jar, give it a swirl to distribute the mycelium and quickly insert the needle.

Tilt the LC jar (keeping the filter patch positioned at 12 o’clock) and begin drawing liquid into the syringe. If it gets plugged, just squirt some liquid back into the jar to clear the clog. Once the syringe is full, quickly remove it and wipe the injection port. Wipe the injection port on your fresh LC jar and quickly insert the needle of your newly loaded syringe. Deposit a few drops of liquid and remove the needle. Wipe the injection port again.

Inoculating Grain Spawn 

Once your grain jars have cooled following sterilization in the pressure cooker, disinfect the entire surface of your grain spawn jar, taking extra care to wipe the injection port. Sterilize your LC syringe before usage, and wipe the syringe and needle with alcohol. Wait for the alcohol to evaporate completely, then flame the needle tip until it glows red. Wait 10-15 seconds for the needle to cool, or dip the tip in a small dish of alcohol. 

Wipe the injection port on your grain spawn jar with alcohol and fully insert your needle, and inject 1.5 – 3 ml of LC. After removing the needle, always wipe the injection port to remove any residue that might attract contaminants, and shake the contents of the jar to distribute the LC. Alternatively, aim the needle at the sides of the jar, and shoot about 0.5 to 1 ml of liquid at each of the four quarters of the jar (using 2-4 ml of LC per jar).

If using grow bags instead of jars for your grain, a similar approach can be used, following aseptic protocol (ensuring there are no drafts, wiping down surfaces with IPA, swabbing, and flaming syringe needle. While some growers recommend performing bag inoculations in front of a laminar flow hood, this approach has been used in the open air with consistent results and very low contamination levels in my experience. Swab the part of the bag you’ll be injecting into with IPA, and flaming and swabbing your syringe needle, inject 5-10ml of LC into the bag. Larger amounts of LC will speed up colonization time, although excess LC may make the grain substrate overly moist, which the mycelium won’t appreciate. Have a few strips of sellotape standing by, and while the syringe needle is still hot from being flamed, inject the LC, withdraw the syringe needle, and immediately seal the hole with the sellotape. Then, agitate the bag’s contents to mix the LC into the grain to encourage even colonization, and incubate at room temperature (or at 70-80F for faster colonization).

Incubating Liquid Culture 

Incubate your jars at room temperature, or 70-80 Fahrenheit, if you seek faster colonization time. Swirl/shake the LC jars at least once every two days to prevent the mycelium from clumping and help oxygenate the LC (this isn’t necessary for the first five days following inoculation). When there is a large jelly-like blob of mycelium filling about 60% of the liquid (approximately 1-2 weeks after inoculation), store it in the refrigerator. Around two weeks following inoculation, a new batch of LC should be sufficiently colonized to begin performing inoculations with it in most cases. When the mycelium has grown and almost fills the jar, vigorously agitate the liquid culture to break the mycelium up as much as possible. At room temperature, colonized LC may retain its viability for 6 months before the LC nutrients are used up, but if refrigerated, it may retain viability for up to two years and may be used over and over again. 

Keep a regular eye on your cultures to check for signs of growth and to monitor the progress. Healthy mycelium will appear as clean white blobs of mycelium floating in suspension. While contamination isn’t always easy to spot in LC, sometimes it will be visible. If the LC liquid becomes cloudy or its color changes and it can’t easily be seen through (judge this about a week after inoculation as sometimes the cloudiness fades), or if there is a greenish layer of scum on the surface after it has been left to settle for a few days, or if the liquid gives off a foul smell, it is contaminated and should be discarded.

Sterilizing Used Syringes/Needles 

Incubating Grain Spawn 

Grain spawn can be incubated at room temperature, although temperatures between 70-80F will speed up colonization time. Various grains can be used, including brown rice, whole oats, rye grain, wheat, whole barley, whole millet, and wild bird seed. Following inoculation with liquid culture, wait until you see streaks of mycelium covering about 15-20% of the visible grains next to the glass, then shake the jar very well to evenly distribute the inoculated grains throughout the jar. This should take about 4-6 days. Alternatively, you may also shake the jar immediately after inoculating it with liquid culture.

Testing LC on Agar 

An optional intermediate step is to test the sterility of your LC on agar, which shows contamination more clearly. Make up some agar in your jars (mixing the ingredients listed below in a pan on the hob briefly), and once the mixture has thickened to about the consistency of runny gravy, add this to your jars (1/8” to 1/4” is a good depth). Sterilize it in your pressure cooker at 15 PSI for 20 minutes. Using a sterile procedure in an aseptic environment, such as in a glove box or in front of a laminar flow-hood, drop a few drops of your LC onto the agar before sealing and incubating the jar.

Agar recipe:

50ml water 1g agar agar powder 1g light malt extract powder (or use 1ml syrup)

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