How To Turn 1cc Of Liquid Culture Syringe Into Unlimited Spawn.

[@ezmushroom]

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[@WhiteBeardHashtag1205]

A couple of notes for those in the comment section. I would only use about half of the malt extract suggested. The amount being used in the video is what I would use for an entire liter pour. I would suggest against the use of red coloring. *Some* red dyes inhibit fungal growth. Note to the content creator I love that youre using Broke Boi jars Have you ever considered getting yourself one of those continuous mist spray bottles for your isopropyl? They work amazingly and use even less liquid. I would also suggest 20mm self healing injection ports for your LC jars. They are super cheap and keep you from having to open your LC jars to draw into a syringe. Nicely done video, and good camera angles. Keep up the good work 😉

[@larrymcfarlandjr2160]

Well done, man. Keeping everything as clean as possible is always a good thing. Mush love!

[@Lemonz1989]

I sterilize my SAB and equipment with a strong UV-C light, so the air inside has less contaminants that may fall into the agar. It works really well. I actually also pressure cook the agar in a bottle and pour the Petri dishes inside the SAB, with the lids off, so I don’t get condensation, and leave everything under the light while it’s cooling down. Then I put the lids on and seal the dishes. I’ve never had a new Petri dish that has been contaminated since I started. I’ve only had 2 contaminated plates when I’ve transferred live tissue samples from the open air to a dish. I turn the light on for 30 minutes and leave the room until it’s done, because it can cause eye damage.

[@user-xb1ht4py2v]

This is the process i am planning on using with one extra LC step. syringe to agar, to LC, to LC to grain spawn. The i have the first LC to use as a mother culture for about 6 months and the initial syringe as a master LC in long term storage.

[@miteshcryptomarathi]

So i get this, master syringe > agar plates > ( grain jars / liquid culture), liquid culture > grain jars. then repeat process to make more liquid culture. So few questions, why not directly master syringe to liquid culture skipping agar plates ? also how do you refresh the master syringe ? also wanted to appreciate the video, always thought flow hoods are must to attain perfection, glad to see it can be achieved using SAB.

[@ezmushroom]

You get to test your syringe for contamination on agar and will know pretty immediately if you failed (within a weekish). If you see contamination you can transfer clean Myc and clean it up. And because you are using a SAB its a good idea to confirm you have a clean culture before going to liquid culture and you start questioning sterile technique instead of a dud syringe or culture. For refreshing, move it to agar then a new LC nutrient source and see if it refreshes. However best thing to do is take a piece of myc and put it in longterm storage on agar / distilled water, sealed. I haven't got a video on this currently but search Edward Grands video as he discusses it and so do others.

[@TheTubejunky]

Tobh LC can still be contaminated even after a successful agar run. You could have likely just got the bit of LC out that wasn't affected. I mean you could just save agar slants just as long so idk. Everyone has a method that works and I say use it.

[@glyndwrevans]

Firstly, nice video 👍 However I'm not getting the step of going to agar first. Put 1ml to 400ml of LC. Then grow that up. Then test your new LC on agar to check for contamination. Then to be absolutely sure when to going to grain, give the LC a good mix and then inject your grain AND onto some agar to check for contamination if you need to. If this method works for you then keep going but it's an extra step that's not needed in my opinion.

[@WhiteBeardHashtag1205]

@@glyndwrevans I disagree vehemently with your assertion that the initial test is unnecessary. You should always test the LC you receive, first, before using it to do anything. Once it tests clean, only THEN should you expand it to properly sterilized LC broth or inoculate grains with it.

[@user-xb1ht4py2v]

@@glyndwrevans that only gives you 10 jars of LC from a purchased master syringe. If you are in production that master syringe will only last you about a month, depending on your production level. With going to agar first you get to test it and also you literally only use a few drops of your master syringe at a time; allowing that syringe to last a few years.

[@edwardgrand]

Nice video. Is that a Feather #7 scalpel handle? I wish I could buy those here :) I have to use the shitty Hilbro ones. LMFAO

[@ezmushroom]

Indeed it is, feels great to work with, slim. I've been watching your videos on serial dilution all weekend man so its great to hear from you, big fan haha! Planning on using it for cordyceps

[@edwardgrand]

Check out the 'grab and drag' stuff. Way easier. I haven't done a serial dilution in over a year. @@ezmushroom

[@captainbonez]

For a hot second there i thought you were pouring tabasco sauce in instead of food colouring 😆 The only 2 things i would possibly suggest with this is, 1) You can squeeze the sides of a syringe quite hard and you can squeeze one drop out, and 2) Long sleeves are prone to contamination Cheers though

[@davegrimmer6725]

Dude where do you get those agar pots from?

[@TeaWhoYou321]

That looks awesome, ive been thinking about doing agar but put it off due to complication but that looks super doable. I am aware of agar plates having a small gap for air exchange and one of your plastic tubs has an air filter made from micropore tape, would you recommend that ?

[@AngelsAckiz]

fantastic video, thank you

[@Naturalpeace7]

Great video keep up!

[@gizmorepairs]

Where did you purchase your still air box ? Great videos I’ve just came across your channel and I have subbed. Thanks for your time and effort 👍🏻

[@ezmushroom]

We sell them on our store, however we are out of stock right now, you can get notified when back in stock ezmushroom.com/shop/mycology-equipment/still-air-box-mycology-mushroom-cultivation/ NorthSpore have a similar product, and there are some on amazon.

[@CasperBenfatah]

i got one on temu for 15£ works great

[@EMDS04]

Did you have any excess water when cooling down the agar ?

[@ezmushroom]

Yes there will be excess inside the container but this clears up after a few days.

[@urszulapiwowarczyk5962]

Thank you for clear explanation how to do this properly. On the other hand… what is this music in the background please?

[@ezmushroom]

Cello Zen - Orchestra/Background/Piano - Royalty Free Music

[@urszulapiwowarczyk5962]

@@ezmushroom thank you so much

[@Brandon-jw5cv]

Weird question but hear me out, can you just do a 20 minute video of stirring the agar asmr-style? 😂

[@NotPaesh]

I'm surprised that heat sterilizing the needle doesn't kill the mycelium inside it.

[@ObscenePlanet]

I would assume the spores inside the metal needle would cook but all the other spores in the syringe should be fine

[@ezmushroom]

This is correct^ Only a tiny amount will be inside the needle which gets pushed out

[@user-xb1ht4py2v]

it does. but it is a tiny amount. It will not kill the entire syringe full, just the tiny bit inside the needle.

[@ianwynne5483]

Do you have a link for those reusable agar pots.

[@ezmushroom]

I just picked those up from Asda in the UK.

[@ianwynne5483]

Thanks, i picked up similar from Aldi, the pots and lids were polyethylene and not polypropylene, they were crushed by the sterilisation process. Lesson learned.

[@TheTubejunky]

​@@ianwynne5483 you can use condiments cups (ketchup cups) from the local box stores just pour them in the SAB. They work fine and maybe have 3 out of 40 or so get contaminated. (Only once was there three usually none when I'm sterile)

[@manuepineda8652]

Heyyy, so, linking the jars video with this one, im a bit confused. I got some preti dishes to do the LME preparation as i got a spore siringe and i want to turn the spore syringe into more liquid culture. Now, when it comes to the Liquid culture jar, do i need to make a hole in it if im going to open the lid and put a drip on it??? I get the reason of making a hole when it comes to jars of grain, as it needs some form of air exchange. But if you're going from agar to LC/Grainspawn inside a SAB by opening the lid with your hands, i dont get the point of making holes in all those jars. Do i still need to make a hole for FAE and an inyection port (for that i bought some red silicone) in both my grain and my LC jars?? Thanks!!!

[@ezmushroom]

If you are going to lift the lid then inject myc culture into liquid culture you dont really need a hole, if you were injecting through an injection port you need a hole as you would need the air pressure in the jar to equalise (LC goes in so Air must come out), otherwise you cant press the plunger down. Also if your using spores go to agar then LC its more consistent than spore to LC (spores are generally not clean). Grain jars require holes for gas exchange. LC doesn't really require the holes as the myc is suspended in water anyway but remember to regularly shake it to mix more oxygen into the watr.

[@manuepineda8652]

@@ezmushroom youre a legend, thanks man. Yep, i will be creating plates of agar with the spore syringe. From there i will do inoculations. Ill probably try all the methods (agar to LC, agar to grain, and lc to grain) see what works best!!

[@gizmorepairs]

What’s the best temperature for growing mycelium in liquid culture and how long until you get a full colony in a 600ml jar ? Thanks

[@WhiteBeardHashtag1205]

74-80f is ideal. If you stir it daily, 10 days should be good, then refrigerate it if you plan on trying to elongate its viability. If you plan on using it all up in the first 6mo or so its fine to leave at room temperature

[@user-xb1ht4py2v]

@@WhiteBeardHashtag1205 Just dont refridgerate Pink Oyster!

[@WhiteBeardHashtag1205]

@@user-xb1ht4py2v I was unaware of this, as I mostly run actives. But still good to know. Thanks for the heads up. But why are you telling me not to refrigerate Pink Oyster LC? What happens to it?

[@user-xb1ht4py2v]

@@WhiteBeardHashtag1205 Pink O. is a hot weather mushroom and temps below about 50F will kill the MYC. Refridgerators keep food below 40F.

[@WhiteBeardHashtag1205]

@@user-xb1ht4py2v noted. Thank you

[@emeteriogomez3748]

How do you go on about maintaining the genetics stable, do you often go back to spore?

[@user-xb1ht4py2v]

going back to spores is the opposite of maintaining stable genetics. It is like going back to seed when you only want to regrow a perfect tomato. Spores, like seeds combine genetics, giving you entirely different phenotypes (varieties) of shrooms. You could clone a shroom or just continue to keep the MYC running. Once you have some jars of LC those will keep for months.

[@theresasteinbacher]

I can't understand what you said that you "plopped" into the LC jar to stir around?

[@ezmushroom]

It was a magnetic stir bar. amzn.to/3X4lYWM

[@theresasteinbacher]

Thank you.

[@AlekssOSZ]

Do you have case of contamination throughout the whole process? Even if you are vert precotionous i wanna Know if contamination is common even if im very careful with sterility

[@ezmushroom]

I didnt get any contamination. If you follow all the steps in this video you should be fine. If anything goes wrong just try again until you get it right its daunting at first but gets easier as with anything.👍

[@mattb6646]

Professional growers and companies consider 10% contamination rate acceptable and good enough to make profit. So if you're 10% or less you're doing good

[@sarahani9303]

Is there an alternative of agar,plz?

[@ФедорБекиров-т9д]

Что означает. ЛЕГКИЙ солодовый экстракт ?

[@fsgrggr5943]

I dont understand... at 11:50 you use the agar plates to transfer the culture to the grain. After that, you also transfer to the liquid solution in order to create a liquid culture. At the end of the video (20:40) you mention however, that you use the liquid culture in order to maje grain spawn. Then why did you transfer culture to grain in the first place? Isn't the step of setting up liquid culture fully unnecessary if you transfer the agar to the grain directly, anyways?

[@ezmushroom]

Yes it is unnecessary if you want to just use agar that is fine. I did this because I wanted to make some grain spawn and instead of exposing the agar to air multiple times I could just do it all in one session (whilst filming). By time my LC is finished, I would also have a few grain jars ready too. The agar can now be stored and I work from the LC jar next time instead.

[@fsgrggr5943]

@@ezmushroom Thanks for the reply, appreciated. But I still don't understand completely. If your goal was to make grain, why would you make syringes before? If I saw the video correctly, the amount of times you exposed the substrate was even lower for agar-to-substrate vs. agar-to-liquid-to-substrate

[@user-xb1ht4py2v]

@@fsgrggr5943 The reason is that you can take a small piece of the colonized agar and create LC with that. You could make easily 10 jars of LC from one plate of agar. Each of those jars can then innoculate 10-20 jars of grain. So from one agar plate you get 100 jars of grain spawn. If you go straight to grain from agar you get maybe 10 jars of grain spawn. So it is only about the numbers. Also he purchased the LC syringes, and hence the desire to keep them long term, by only using a few drops at a time, in order to maintain the genetics.

[@theresasteinbacher]

Could you give me the link to those cute little petri dishes that can be sterilized? Thanks.

[@ezmushroom]

amzn.to/45bjuYN

[@theresasteinbacher]

@@ezmushroom Thank you so much!

[@goazu]

Where do you buy the jars?

[@ezmushroom]

I don't buy Jars. Buy Beetroot, Pickled food, Saukraut, etc. I've collected most my Jars over years. The lids are often interchangeable and if they break they were going in the bin anyway so nothing is lost.

[@goazu]

@@ezmushroom Thank you! Can I use a couple of drops into the agar, once the agar is colonized, could I do agar to agar?

[@ezmushroom]

@@goazu Yes definitely.

[@theresasteinbacher]

Do I need a “stirring plate”?

[@ezmushroom]

No it just speeds up the process

[@theresasteinbacher]

Thank you for your prompt reply. How long does it take for this process without the stirring plate and do I physically shake the container?

[@ezmushroom]

Probably 5 days faster. My advice is just to give it a shake it daily, lightly. You dont want the LC going over the lid and possible seeping out etc. Alternatively you can leave it to just clump, then when you go to draw it up, pump the syringe which will break it up. Its probably worth me doing a video outlying this soon as I never used to use a stir plate. Ill put it in backlog👍🏻

[@ugorjinelson]

Where did you get liquid culture

[@brescalofrio1]

i cant understand english. i feel really dumb or i need to sleep.. the most sterile is the outside edge or the center of the agar

[@ezmushroom]

Take samples between the center and the edge.

[@brescalofrio1]

@@ezmushroom ohhhhh so never go too close to the edge or the center where you droped it... that means i get the growing ends of the hyphae genius

[@user-xb1ht4py2v]

@@brescalofrio1 pretty much. You want to get the growing edge of the MYC mass, but do it before it reaches the edge of the pertri dish. The dishes allow a bit of airflow and that happens at the edge. So any contamination that happens during the growth period will usually happen near the edges,hence the reason to avoid them.

[@AAAA-bk7kq]

[ I wonder whats all this sterile working is all about, because how would they have survived in nature then where NOTHING is sterile, anyone thaught about that? Where are these found in the wild then?]

[@ezmushroom]

Its basically just luck, millions/trillions of spores are released, very few become mycelium/mushrooms in fact majority of them don't. Its similar to human reproduction, millions of sperm cells released but only one creates a human. However if you were to go for IVF treatment they'd want to increase the chance for success therefore they use 1 + 1 under sterile conditions wherein competitors don't exist.