The Agar Sandwich Method | Eliminating Bacterial Contamination from a Mushroom Culture

The agar sandwich method is a great way to remove bacteria from a mushroom culture on agar plates. The method is pretty straight forward, just pour warm sterilized agar overtop of the contaminated agar culture you are trying to clean. The mycelium will grow through the top layer of agar but the bacteria will stay in the middle layer. This leaves us the opportunity to transfer the surface mycelium to a new plate ensuring the removal of bacteria.
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[@FeatherzMcGraw]

Very cool, not seen this mentioned anywhere in my research!

[@shroomboomroom]

It's called cabin sequencing

[@WhiteBeardHashtag1205]

me either

[@WhiteBeardHashtag1205]

@@shroomboomroom no shit? Ill have to check this out

[@ellzey312]

Same

[@WanderingMindful]

A pretty improved variation of cabin sequestring. I mean the only thing is he should have included a warning about pouring the agar too hot ...​@@shroomboomroom

[@Microbe_Master]

You’ve earned my subscription by showing me this.

[@SporenSprout]

Thanks for watching! 🙏 🍄

[@MoreTuck]

same

[@archiewebster5034]

This is a great method! + you could even give the mycelium a better chance at escaping from the bacteria by pouring the second agar layer with a high carbon, low nutrient agar mix.

[@janespright]

Criminally underrated channel. I've seen larger channels teaching just 20% of what you are showing and having more members. Thank you so much

[@pyrocircus129]

Pro tip! First time I have ever heard of this, thank you!

[@WhiteBeardHashtag1205]

this is fascinating! I have never once heard of this method, but it sure sounds legit AF! I rarely have tams, but the very next time I do I am definitely going to try this method 100% I love when I come across a new method Ive never seen that looks legit. Even as old as I am, and as long as Ive been doing this, there is ALWAYS something new to learn. Spore and Sprout, good job making this video and bringing info to people that wouldnt have found it otherwise. You earned a subscription from me

[@SporenSprout]

Thanks for the kind words! Much appreciated 🙏 Thank you for watching! 🍄

[@skrazi]

Thanks for also being a valuable member of our myco community, you are noticed 🍄 I've heard of this method before but spore and sprout always makes it super easy to follow.

[@WhiteBeardHashtag1205]

@@skrazi I appreciate your kind words and candor. Were all out here trying to help beginners get started. He even taught this old dog a new trick, lol! I am new to Spore and Sprouts channel, and I look forward to watching some more of his videos

[@taskos1261]

Finally someone did it. Fantastic job mate. I am struggling through ages with embedded bacteria especially with wild hericiums. Now its time to shine. I wonder if wages of agar would have the same result, in case one could avoid pouring etc.

[@SporenSprout]

Yes a square wedge can work also. I’m gonna make a video about that method as well. Thanks for the support and for watching! 🍄 Good luck!

[@huntingicon]

That is the coolest thing ever! Thanks for sharing

[@SporenSprout]

I agree It is so cool! 🍄 Thanks for watching!

[@benchadwick1754]

Well that, sir, is amazing.

[@SporenSprout]

It’s really cool! 🍄

[@coffeyscreations4342]

Very interesting!! Thank you for sharing

[@SporenSprout]

Thank you brother! Everyone go subscribe to this guy btw

[@__shaun__]

nice work mate!

[@altaran]

You didn't mention, but whats the max temp your agar should be before pouring over the mycelium/contam?

[@SporenSprout]

125 F is good.

[@altaran]

@@SporenSprout Thank you!. :-)

[@RichardBronosky]

This is very close to the 130⁰ drying temperature that I'm told renders dried mushrooms inert. Do you agree with those values? (125⁰ = safe; 130⁰ = inert)

[@altaran]

@@RichardBronosky Nahh, i think your numbers are wrong. Its closer to 200f before they start breaking down. Dont have the exact number atm.

[@malekith8215]

Thank you very much . Good question. I was just about to try that method and asked myself the same question. Also thanks to spore and sprout for the quick answer 😊

[@jasonmillar2626]

very cleaver bro, thanks for the tip

[@nittygrittykitchen]

You are innovative! Thank you!

[@chaseTheCase9]

No way! Awesome 🤘🏼

[@MrZensphere]

Fantastic tek, thank you!

[@SporenSprout]

Thanks for watching! 🍄

[@ibberz]

I have a few bacterial plates to clean up! Definitely giving this a try

[@klaushermann6760]

Blessed. 🙏

[@ObakuZenCenter]

Great video. Thank you.

[@jonathanscrocco1320]

o wow..genius dude! beats me always trying to save a corner

[@reaganadams379]

Ive heard of flipping a plate to take a sample from underneath the agar plate but never tried it. This is amazing. Definitely will try with my wild Hericium Coralloides transfers to save a tonne of plates.

[@SporenSprout]

Yeah that is another cool method!

[@nakedoaktreefarm]

This is genius

[@SporenSprout]

Thanks for watching!

[@Mr.Gandy.]

vielen .dank.

[@D0CT0R420]

Very interesting. I will have to give this a shot

[@goatrock123]

Nice! Never saw this before. Like using chopsticks.

[@sterher]

Nice work as usual. I used to transfer bacteria cultures using flame sterilized wire loops. They were so thin you didn't have to wait for them to cool. They were fine to use seconds after heating.

[@SporenSprout]

That’s awesome! The tools I use are really thick so it takes a while 😆

[@MissBlackMetal]

Why did you stop using them though?

[@SporenSprout]

@@MissBlackMetal He was a science teacher in school.

[@MissBlackMetal]

@@SporenSprout gotcha, so it isn't that he stopped liking the wire loops in the end, or anything like that. That's what I wanted to know -- thanks! I'll look into a wire loop as a secondary tool 🤘🏻

[@derrickcarroll4932]

cant wait to try this!!!!

[@rgarden-of6hf]

Would reversing a plate be a reasonable option? Perform as normal. After agar is hardened Using a plate cover with agar hardened on it to close up. Wax tape. Let culture grow. Potentially crawl to top plate?

[@RebelionFiscal]

Nice video, very well explained. I suppose this method is for when water agar doesn't work... Right?

[@gabet7333]

Thank you for sharing this

[@SporenSprout]

Thanks for watching! 🍄

[@YukonKornelius-ur4xw]

I thought the hot agar would kill the mycelium but I guess not! I've used water agar to clean up cultures before but never even thought about this method. Nice work, man!

[@SporenSprout]

Yeah the hot agar doesn’t even phase the mycelium. I like working with water agar too, always room for another cool method! 🍄

[@WhiteBeardHashtag1205]

Agar liquifies at under 100f, and it cools pretty quickly. I think it might stunt the mycelial growth momentarily, but I doubt highly it will kill it. If it did, this method would not have the results it does.

[@ricklulu3572]

I am pretty sure that it does kill the mycelium and the bacteria, but only on the top of layer. That's because the reason why the mycelium has embedded itself way deeper down. I am just theorizing but Im sure iam right.

[@SporenSprout]

@@ricklulu3572 No it doesn’t kill anything.

[@WhiteBeardHashtag1205]

@@ricklulu3572 if youre just theorizing, then why are you sure youre right? Im so confused...

[@ricklulu3572]

I guess the heat does kill the bacteria but does not kill the mycelium because it is embedded deeper down.Thank you for this idea.I will use this idea if I need to

[@StealthySpores]

This is amazing!!!

[@samuelgeorge8524]

This is a really cool method that I've never come across! Thank you for sharing. This can really be so damn helpful! Cheers!

[@SporenSprout]

Thanks for watching! 🍄

[@malekith8215]

Paul stamets describes This method in his book “growing gourmet and medical mushrooms” , but he mixes antibiotics like gentamycin sulfate into the second agar layer. I would be really happy if it works just with the warm agar like you describe it. I don’t want those antibiotics in my shrooms.

[@svenwaddell]

tattoo magnum shading needles work great for transferring mycelium.

[@GrowYourOwnFun]

Super cool!!

[@SporenSprout]

Thanks for watching! 🍄

[@bridgesjohns6492]

genius

[@MacCelium]

This is a brilliant technique. I'll put it to use next time I see some strange behavior on a plate. Also was wondering about your 75 degree incubation temp. I always leaned towards about 80. Have you tried both? Thank you!

[@SporenSprout]

Yeah anywhere between 75-80 F is the sweet spot.

[@SilverPhoenix-PlazmaMoon]

dope af

[@YaBoiii666]

nice filming

[@Onthruthestorm]

Thank you!

[@SporenSprout]

Thanks for watching! 🍄

[@DavidTheHappenings]

This is genius! Are you using standard MEA for the top layer or plain agar with no nutrients in it?

[@SporenSprout]

I just used left over nutrient agar but you can use water agar too.

[@chicofromph33nix64]

Amazing job dude... never seen this but seems legit... I use ketchup cups and wondering if this will still work the same? I don't see why it wouldn't..

[@Andrea-yo3oe]

🔥

[@StickyDankFingers]

Outstanding solution :) Will this method work for what I believe is a yeast “infection” on the agar surface as well. I think it’s yeast because it doesn’t look like any of the typical contaminations

[@SporenSprout]

Yes it should work!

[@StickyDankFingers]

If you should ever get yeast would you be kind enough to show what it looks like. Does yeast come from condensation ? Thank you

[@greenthumb42_56]

I’m trying to cultivate shiitake mycelium from dried store bought mushrooms I’m struggling to create contamination free mycelium. Could I place a dried mushroom into a Petri dish and pour agar directly over and get similar results? or do I need to do the initial Petri dish culture followed by a pour over “agar sandwich”? Thanks for the video! Always thought a contaminated Petri was a lost cause. New found hope!

[@SporenSprout]

@@greenthumb42_56 Check out this video: kzbin.info/www/bejne/i5eXpoWvp653sM0

[@kikimusa685]

I wonder if this would work if the mycelium hasn't even taken hold yet?

[@Iamfarashuu]

What temperature is the new agar that is used to sandwich the bacteria? I should have read the comments.lol Great info

[@biswakarmababooraam3823]

❤

[@jimycrackcorn408]

What is the right temperature to pour the liquid agar on top of your plate like what is too hot and what is effective and efficient?

[@SporenSprout]

120-130F is good

[@jimycrackcorn408]

@@SporenSprout thank you very much. I appreciate the response. Love the video. Hopefully this solves some of my issues. No I just need to figure out the most effective way to turn my agar plates into LC's. I've never been able to transfer agar to grain and have it not get contaminated ! So I would like to turn my plates and LC for faster colonization that has also been a difficult process for me!

[@jimycrackcorn408]

@@SporenSprout do you have an opinion on using antibiotics in your agar?

[@SporenSprout]

@@jimycrackcorn408 You could buy this cheap mini flow hood to make the transfer from agar to LC: kzbin.infoDCLCM5r39IY?feature=share I have never used antibiotics.

[@ObakuZenCenter]

@@jimycrackcorn408 If you use a still air box, have the arm holes covered and let the box sit for about an hour, after putting all you need in there, there should be less contamination.

[@jacobgarcia3597]

so this is a method to get rido of embedded bacteria?? cool

[@SporenSprout]

Yes!

[@tttiiooo]

I kinda want to contam one of purpose to try this.

[@SporenSprout]

😂 in the name of science! 🧫 🧬 🍄

[@HighTierScrub]

Why can't the bacteria come up the agar again? Are they slower than the mycelia???

[@SporenSprout]

Yes, the mycelium wants fresh air at the surface more than the bacteria does.

[@garrisonalan8842]

Wondering where you got your rack that is in front of your flow hood I can’t seem to find one like that

[@texaspowerman]

Interesting, any idea why the bacteria doesn’t ride along?

[@SporenSprout]

The mycelium loves air at the surface more than bacteria does.

[@texaspowerman]

@@SporenSprout I know there are anaerobic bacteria, but are those growing on top of the plate that you cover up? I’m going to have to poke at this a bit and see what I can dig up, so to speak. Very curious idea. I toss to many plates because of rotting gill fragments on the swabs I get. Some people look like they used the swab like a shovel. ✌️

[@BeefSupreme-vk9sz]

I've done this then.....fruited them and get a good amount of shrooms, the prob is the agar dried out a lil quick and doesn't take on water 😂

[@breik8183]

Gawd i love youtube

[@Awakenedsoul2024]

Would this work against slime mold?

[@ObakuZenCenter]

Worth a try but best to use this for bacterial contamination, rather than mold. Agar is cheap so as long as you're using a decent still air box, you should be able to get enough clean mycelium to do good plates.

[@RichardBronosky]

🏆

[@RichardBronosky]

Can you recycle the contaminated agar?

[@ObakuZenCenter]

It's best not to. Plus, agar is so cheap there is no need to.

[@ElvinHernandez]

Temp of agar?

[@SporenSprout]

It was around 120F

[@ElvinHernandez]

@@SporenSprout thanks!!

[@CarlKeeling1881]

My cordyceps militaris got contaminated 😔

[@SporenSprout]

What color was the contam?

[@CarlKeeling1881]

@@SporenSprout green

[@SporenSprout]

@@CarlKeeling1881 Dang that sucks. It’s usually from environmental conditions

[@CarlKeeling1881]

@@SporenSprout yeah most likely I really need to make a flow hood. Have you diy-ed a flow hood?

[@SporenSprout]

@@CarlKeeling1881 Yeah I have made 2. Here are the 2 components that you can build the box around. 1. HEPA filter: amzn.to/3xl5qPr 2. Blower motor: amzn.to/3U2d36l

[@BadddDoggg-id4po]

FAR OUT!