iquid culture or LC as it’s often referred too is mycelium suspended in a nutrient broth. The purpose of LC is to make inoculating substrates and grain spawn easier. The risk of contamination is lower because the culture goes from a sterile environment (Inside the jar) to the sterile syringe and is injected into the new, sterile environment rather than transferring with agar wedges, increasing the risk of contamination, especially to more inexperienced growers.
LC does have some downsides, because it doesn’t grow on a ‘horizontal plane’ like agar does, contamination can hide in and amongst the suspension, only showing itself once the culture is dropped into a new environment, this is why working as clean as possible with your culture and LC is very important.
Once you have a bottle of sterilised ‘broth’ you drop a sample of your desired culture into it. Within 7 – 14 days you’ll see the mycelium has ‘clouded’ the culture and expanded throughout; this is aided with semi-regular mixing/shaking. This can be achieved by use of a magnetic stirrer, a bolt in the bottom or just swirling by hand.
Things you’ll need:
AirPort Jar – Here is my guide on how to make them.
Syringes and Needles – From My Shop
Malt Extract – From My Shop
Firstly, dissolve 20g of LME into 1 litre of water, I tend to this with hot/boiling water as it helps dissolve it better.
Fill your jars/jar approximately 80% full with broth.
If you have a magnetic stirrer add it now, if not, add a bolt. If you don’t have a bolt, don’t worry you’ll just have to swirl a bit more. Place your broth into the PC and cook for 15 minutes at 15psi. Cover the tops in tin foil to protect the filter.
Wait for the PC to naturally vent pressure. Forcing the PC to a lower pressure by removing the rocker or opening the valve will cause your broth to overboil and push up through your filter, making a right mess. Once the PC is at zero, remove the jar and wait for your broth to reach room temperature, anything too high will stunt/kill the culture so be patient.
Prepare your area under the sterile working condition of your choice i.e. SAB or Flowhood, all surfaces rubbed down with 70% Isopropyl Alcohol including the jar, flame lamp for the scalpel etc…
Transfer a small wedge, roughly the size of a 1st class stamp to your jar.
Seal each jar TIGHT and add some micropore tape/parafilm around it for good measure.
Incubate at the appropriate temperature for your species, I’m using blue oyster so I will incubate at about 24c. Wait 7 – 14 days to allow for colonisation, once you begin to see growth, give the jar a gentle swirl/stir and repeat as needed to break up the structures and ensure the whole broth is colonised.
This step is optional, stir/swirl your culture and take a sample by drawing it into a sterile syringe with a heat-treated needle through the injection port and dropping a single drop onto agar and allow the mycelium time to colonise
If your sample is clear and it colonises normally without contamination, continue using it in the knowledge your LC is clean and ready to use!
A lot of work goes into these blogs, if it helped you out and you can afford it, then you can support my work by buying me a pint! Cheers!
Hi there,
I’ve been reading your blog and find it quite useful for growing mushrooms, I have a question and hope you can help. I created a maitake mushroom liquid culture from a syringe, mason jars with ports into sugary water. The culture looks healthy but I’m wondering how long it will last for before I inject in into grains. Can I refrigerate it if I don’t use it all on the grain? I started the process on August 8th.
Thanks!
Sarah
Hi there Sarah!
That’s great! You can fridge it for a good long while i’d say upwards of 6 months, though it’s potency and vigour will diminish every day. It will continue to grow slowly in the fridge and can ‘choke’ itself my making a mycelium raft. Storing on agar or in a master slant is much better as you can just throw it in the fridge and leave it. With the LC, you’ll need to stir it fairly regularly to avoid it all fusing together. Liquid culture is a bit of a pain because it can hide contamination very easily. Healthy LC may hold contamination and you won’t know until you put it on grain or, much better, is to test it on agar!
My advice, drop some onto agar and keep it that way for around 6 months before it begins to dry out!
Good luck with it though and good luck with growing Maitake!
All the best!
Gareth
hi, when you wrote “transfer a small wedge, roughly size of 1st class stamp to your jar”, what is the wedge? is it a sample of the type of mushroom you wish to grow?
thanks
Hi mate,
Thanks for the comment! When I talk about a ‘wedge’ it means a wedge of agar. That is the mycelium of the mushroom you want to grow that has been grown out on a nutrient rich medium, agar! Hope that helps chap!
Cheers!
Gareth
Hi!
Thank you for this information. I plan to follow these steps later today. I just had a thought. What if one were to drop multiple types of wedges in the jar? For example Blue Oyster and Lion’s Mane. Would it create a hybrid? Thanks!
Hi Ana!
Thanks for the question, no it wouldn’t work, they would just compete for resources and the victor would be the winner! Since Mycelium is the mated part of the fungus, you’d have to go and look at single spore isolation and cross breeding! It’s a very complex science that I have no clue about!
best of luck!
Gareth
Hi Gareth,
First of all thanks for all the information you share
I was reading your article and I have a question on the quantity of the LME.
You mention 20g LME per 1000ml. I use ~1g per 600ml from what I have researched online.
Is there a reason you use so much more?
Im new to this so might be im missing something.
Thanks a lot
Hi Kostas!
20g Per 1000ml is a fairly common amount for LME Agar. You can run it leaner or richer to achieve different results. The leaner the mixture, the less nutrients the myc will have so it will ‘strech’ as Rhizomorphic growth. Using such a small amount has it’s benefits especially when cleaning up cultures.
I can’t say why that’s the ‘common amount’. With some digging i’m sure there is a scientific explanation as to why, but I know that amount has always worked well for me! If I were you, i’d run a comparison with the same culture on differing Agars and see what results you find!
Good luck!
Cheers,
Gareth
I have a few questions about liquid culture. Would it be ok to strain. I used light malt extract and it is very cloudy. At what point should I transfer to a plate to test for contamination. I have used kayro and one for the two different liquid culture I am using. What would be a better recipe I want to try to gain as much experience as possible. One more question when starting from spores how should I go to liquid culture from syringe to agar to liquid culture or directly to liquid culture.
Hi mate,
Malt isn’t a clear as Kayro or Honey. But there is really not a great deal in it when using malt vs dextrose vs honey. Lots of different recipes and many people have their preferred LC recipe or one they’ve seen improvement with a certain strain but i’ve never noticed any variations or improvements between different LC’s. Malt, however, has a tendency to have bits that float around inside the LC. They’re just burnt sugars and absolutely nothing unusual or worth worrying about!
Depending on the species and strain, anywhere from 2 – 7 days before testing, just make sure you mix it thoroughly either with a stirrer bar or something like that!
I would always test your spores and LC first. I even transfer any cultures on agar once or twice to make sure nothing is hiding. Just because it’s not blindingly obvious, doesn’t mean the culture is guaranteed clean!
Always test! It saves energy, time and resources in the long run. I’ve wasted a lot of grain learning this!
Cheers for the questions! All the best!
Gareth
Thanks for the info, much appreciated. Being a novice, I’ve a few queries.
Instead of using a ‘wedge’, could I use a commercially purchased liquid culture syringe and expand this out into a jar?
What is the best medium to store liquid culture on for several months?
Cheers!
Hi mate,
You absolutely can, just test your LC on agar first, even from a commercial producer, LC still hides nasty piggy-backers! The best way to store cultures are on ‘Slants’ LC can strangle itself, even in a fridge, after a few months. It begins to form rafts ontop of the LC and eventually dies because of poor gas exchange and the build up of it’s own waste products in the LC. I wouldn’t store a culture I really like on LC for extended periods of time!
Best of luck!
Cheers,
Gareth
Hi, thanks for the info!
Just wondering if it would be possible to transfer a bought LC in to zelf made broth.
Than keep 1 jar of broth+LC in the fridge and use this for a couple of months to inoculate new broths (and grains later on).
Would is be safe when using a lid with injection port?
Mabe testing it every now and then on agar?
I’m just looking for a low tech solution to keep a LC without a flowhood and lab.
Regards, Bas
Hi Bas,
If I understand what you’re asking ‘Is it safe to keep LC in the fridge?’ The answer is yes, depending on the species. I’ve had LC in the fridge for 6 months and it’s still been viable. You can keep taking from it but every time you cool mycelium you’re sort of degrading it so you’d need to work relatively fast by dropping it into the new broth. Don’t forget, every time you use your syringe, you may risk contamination hanging around the tip, especially with LC since it’s mostly sugar water!
It would be safe to use an injection port, however, over time and repeated uses the ports do wear out. Slants are a safe way to keep culture viable for long periods of time. you can just use a needle to take a biopsy of your slant and drop that into your LC.
All this can be done in a SAB!
Hope that helps mate!
All the best,
Gareth
Hi Gareth,
Thanks for your reply.
I’m not looking to keep a culture for more than a year as I’m willing to buy it once a year if needed.
I’m really looking for a low tech way to make grainspwan with LC. I was thinking to buy a LC, than inject it in to zelf made broth (jar with injection port). And then every 3-4 weeks I’ll transfer 9/10 in to sterilised bags of grain and 1/10 in to a new zelf made broth to keep for the next round. It seems like an easy way to keep a culture for some time and an easy way to make grainspawn. I’m just not shure if this wil work and if it’s more risky for contamination. I can change the injection ports every now and then and use a new sterile needle if needed.
Thanks for the help mate!
Regards, Bas
It will work mate, there are always vectors for contamination and LC is not bulletproof.
I’d suggest you make a master slant and take from this, that way there is little to no degradation of the culture as you’re always taking from the master and not perpetually cloning clones. Agar work isn’t really a step up from regular LC work, especially with the use of a SAB. I’d suggest joining ‘Agar of Asgard’ on facebook, there are some awesome agar plates made on there and the majority are made in a SAB!
Best of luck mate, let me know how you get one!
All the best,
Gareth
Hi, can you explain what the syringe filter is for and how it is used please?
Hi Dave!
It’s a plastic, sealed port, that contains a 0.22 micron piece of filter paper. This is use as a means of sterile air exchange since mould spores and bacteria generally cannot get through! Mycelium intake O2 and exhale CO2 so need to have a way of breathing without exposing the sterile substrate to contamination!
I hope this helps!
Thanks for your comment,
All the best!
Gareth
Hi Gareth when you say slants do you mean peatry dishes ?
Hi Jamie,
No mate, slants are essentially test tube shaped vials that are set at an angle of between 15 – 45 degrees. They usually have some kind of wood in the agar too. The small surface area and nutrition mean you can maintain the culture in cold storage for a long time without drying out. Some claim up to 10 years and the culture is still valid. Long term storage on a petri dish will result in the culture drying out.
Cheers,
Gareth
Can I make liquid culture ,using malt extract agar , since I have it, and if yes ,at what ratio?
Thanks
Hi mate,
You can make LC using Malt extract but don’t use agar or it will solidify. 20g to 1L of water.
Best of luck!
Gareth
Howdy! So, spore syringe to liquid culture – how long should it take for mycelium to form? How can I tell if the LC has gone bad…? Thanks!
Hi Jim,
I wouldn’t go straight from spores to LC. Spores aren’t always caught in a sterile way. I would go Spores – Agar – LC – Agar (test) – Inoculate. Under optimal conditions you should have growth within a week or so.
All the best,
Gareth
What is the best way to isolate pure strain from mushroom.
Hi mate – You’d need to isolate spores and match them to breed the desirable characteristics. It’s a long process because you first need to isolate, then match, then breed and see the results.
Cheers,
Gareth
Greetings Gareth,
Great source of information on here, thank you for sharing. Question, I’ve used liquid cultures with 4% honey and they’ve worked out well. I’ve also used LME (from Wilko) at a ratio of 2 grams per liter (I know you use 10 times that, ie 20 grams) and 1 gram of peptone. Both LC’s have worked well on all species so far except one: Lions Mane! It does grow but very slowly compared to all my other mushrooms LC’s. I’ve read somewhere that Lions Mane requires a “special” formula, any idea? Or is it normal that Lions Mane is such a slow grower?
Hi there!
Good to hear you’re getting into it! Lions mane grows much whisperer than other species, so though they may not look as dense, the mycelium is still there! They will thicken out over time but there is no ‘one’ recipe they favour!
All the best,
Gareth
Thanks for this post! Should my LCs eventually take up the whole jar? No signs of contamination and pretty rich when using a stir plate. But after stir, the blob takes about an inch at the bottom. Was hoping for continuous growth. Vigorous, but no noticeable growth in mass after a month or two, kept in a chamber between 75-82 degrees, covered. Thanks!
Hi Eric!
They should take up a large portion of the jar once you’ve stirred them, however, it’s not essential that they’re ‘so dense you cant see through them’ Evenly broken up strands are whats more important! THe smaller the strands of myc the better as they have greater surface area!
How is your air being exchanged?
All the best,
Gareth
Hello,
I don’t have a pressure cooker and wanted to try out making a liquid culture, is there another way to sterilise the liquid sugar mix before adding the mycelium I wish to make a liquid culture from? And would I leave the modified lid on to sterilise that too or sterilise it another way? If it goes well I may invest in a pressure cooker later but I didn’t want to spend out until I’ve tried it out.
Cheers
Adam
Hi Adam,
Unfortunately there isn’t really a way of doing it outside of a pressurised environment. I’ve heard of people using their oven but not seen much reported successes. We use pressure cookers because the increase in pressure raises the boiling point of water vs an oven which the water will remain at 100c and just boil off, even if the oven is on 200c. If that makes sense.
You can find some absolute bargain PC’s on Facebook/Gumtree market places. You don’t need a presto/AA or anything fancy. Most of us start out with a £20 jobby from Argos!
All the best,
Gareth