How to Make Plates, Slants, and Liquid Culture for Mushroom Cultivation | Agar Recipe | Lab Skills
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- čas přidán 27. 06. 2024
- Mycology Lab Supplies: bit.ly/3mZTUUs
In this video, learn how to make plates, slants, and liquid culture for mushroom cultivation & preservation with North Spore’s head mycologist, Jon Carver! He’ll show you how to prepare three different types of media typically found in mycological settings. This includes MYA (malt, yeast, agar), PDA (potato, dextrose, agar) and media for liquid culture. Included in the video are recipes for agar preparation and liquid culture media.
These are simple, universal recipes that can be expanded on depending on the application. Although we shot this video in the North Spore lab, many of these techniques can be adapted to home or low-tech environments.
For more information on these processes including a complete list of lab materials, ingredients and low-tech alternatives, click the link below!
Making Plates, Slants, and Liquid Culture Blog: northspore.com/blogs/the-blac...
Liquid Culture Multiplier Kit: northspore.com/products/liqui...
Mycology Lab Starter Set Kit + NocBox Still Air Box:
northspore.com/products/mycol...
Pre-made Agar Plates:
northspore.com/collections/la...
Pre-made Agar Slants:
northspore.com/collections/la...
North Spore Culture Bank:
northspore.com/collections/cu...
All American 75X Electric Steam Autoclave Sterilizer: northspore.com/collections/st...
Thanks for watching! Let us know in the comments what you want to see next!
Index
00:00 - Introduction
00:27 - Liquid vs solid media
02:50 - PDA, MYA, and liquid media
03:34 - How to make liquid culture media
04:08 - How to make MYA for plates
04:46 - How to make PDA for slants
05:45 - How to prepare slants for the pressure cooker
06:50 - Pressure cooking media
08:26 - Working in the lab
09:11 - Creating the "slant" in slants and pouring plates
13:31 - Using culture media
13:51 - How to inoculate an agar plate using a slant
14:29 - How to inoculate a slant using an agar plate
15:03 - How to inoculate liquid culture using solid media
15:20 - How to inoculate liquid culture using a culture syringe
Music
“Even Though it Hurts” (instrumental version) - spring gang
“Principles” - _91nova
“I Should Let Go” - Rambutan
“By The Palms” - Gregory David
“Dreamaway” - dream
“Pickup Sticks” (instrumental version) - Pandaraps
“Bringing Me Down” - Ooyy - Věda a technologie
I love pouring my plates. I have actually found a surprisingly high success rate with open air pours (approx. 80%) and one of the big choices I've made to increase my yield of health plates is accept the condensation and pour quite hot. It's hard to handle, and the condensation can be frustrating at times, but accepting the limitations of my work space has really helped to make me quite happy with it.
Great video and it's really nice to be able to check and grow my lab skills with an organization like North Spore Taking the lead.
Thanks for the kind words Matthew! Great to hear you've been having success with open air pours. We're hoping this video helps people feel more comfortable working with media. I think many people are intimidated by the prospect of having contaminated plates, but really mushroom cultivation is an odds game. The more you control your environment the higher your success rate will be, but that doesn't mean you can't do the same process with high success rates (like your 80%) outside of a laboratory!
Wear a Kevlar cut glove and put a latex glove over it. It will help you easily handle hotter temperatures and could be a cheap way to further increase your success rate.
I just pour my petri dishes before sterilising them. I get a few drops on the lid but i know it's clean.
@@chrisakaschulbus4903 I was wondering about that!!
I'm VERY new, though. No experience, except kits. I'd like to expand, and am just trying to "absorb" 😅
I have had a lot more success with a still air box than open air but if I have a plate that's already contaminated with yeast or bacteria I get a spray bottle with 3% peroxide and wash the contaminants off with it in open air, if it's mold I put a drop of alcohol on the contaminant before cutting it out. I've found that with fully colonized plates the chance of contamination is very low even when exposed to open air. Basically bare agar is highly susceptible to contamination but mycelium is not.
That is an extremely informative and professionally produced video. Outstanding in every aspect, camera shots, lighting, audio, The whole deal. Very impressive.
Wow this is the most all around informative video I've seen on these specific methods. Thanks to all involved in the production effort and at North Spore!
Just ordered a martha tent from your company. These videos are very helpful and made me feel confident enough to try growing mushrooms.
Very cool and such an awesome knowledge. North Spore videos are my happy time. can’t wait to put this knowledge to use. 🙏
The best and complete video so far! Thank you
We're glad you enjoyed it! Mush love!
Wow what a great video, thanks for doing a video with all these different media! ⚛️
Nice video, nice editing, it was pleasure to watch!
Very good and well put together video about all of this wow .
So well done!!
Great stuff guys. Thank you.
Our pleasure!
Great video thank u so much
Interesting and thorough. Thanks for sharing
Thanks for watching!
Well done, thank you. Liked and subbed.
Or I could store dry spores in little glass bottles and feel like a wizard.
I'm wit u
😂
But spores do have a genetic variation that cultures from a cloned genetic don’t have
Yes
@@antonetwas2415that’s part of the magic
I think is my favorite hobby thus far as is adorable in my fish bowl much like succulents blessed ✨
Thanks for the tutorial.
You bet!
Great video
Amazing.. 😊
fancy for sure
This video is significantly informative for how short it is!
Also, is there a link for that flowhood??
I just got a bunch of stuff for my first grow, built my own still air box, and gotta get my fruiting mono tub ready to go. I did order an i collated grain spawn of lions mane to start with and I’m hoping I can successfully make some agar plates for the future. I’m really nervous for some reason lol
Take a starlized bit of wood (i use popsicle sticks) and put a bit of it in your slants and the mycelium with colonize the wood and you can clip a bit off it when you need it.
Thanks, good stuff. I've always appreciated your professional approach and production value. There is something fun and rewarding looking at stacks of freshly poured plates. Kinda geeky for sure, but fun none the less. A question if you have a minute. Also, I have some autoclavable petri dishes but no flow hood. Is there any problem doing a no-pour technique (pouring the plates before the pressure cooker), bagging the plates in an autoclavable bag and then doing a PC run? Thanks you.
In the spirit of passing on knowledge from others a couple kitchen tek tips for viewers. Regarding condensation, if you put a mug of boiling water on the top of the plate stack and let it cool completely (make sure it is as wide as your plates) it will often clear the entire stack of most condensation. (I've even used a porcelain baking dish for a couple dozen plates at a time). The oven is a good place to let it cool (just be sure to put a warning note on the control panel :) Regarding open air pouring: What is called Oven Tek has worked well for me on many dozens of plates. You can look it up, but basically you use a warm oven to create a Bunson Burner effect (warm air rising) by pouring in front of the open-door oven. You can use the oven door as a worksurface or, like I do, I use a top rack with a cutting board wrapped in foil with the oven door only open part way. Everyone's environment is different when it comes to airborne contam, but it is an option, and in my (and many others) experience, it outperforms open air.
On the topic of environments. When you are getting started with agar..which you should, it is helpful to get a baseline of your workspace. Getting some pre-poured plates like the ones from NorthSpore, and using a couple to assess your environment can help. Try leaving then open for 30 seconds or so in your potential workspace and then store them in a warm place (80 degrees or so) for a few days to see if anything grows, While certainly not perfect, it has saved some of my students a ton of time and frustration finding out that they have a very high contamination risk. Mush luck!
hey great video! are you guys using nutritional yeast?
Wow mind blowing information about science of mushroom make 1 video about every equipment you use so all understand about equipment & future investment or how to use etc thankyou so much it's really good easy explaining make more videos so knowledge can spread thanks
Man your agar & LC came out so nice & clear, did you filter them ? ..when I use malt and or yeast its always pretty cloudy, The LC will clear up once mycelium starts to colonize it though, but man your media is clear, nice lab you have there too. Thanks the the video !
I love pouring plates lol. I dont know why I just have a great time making my Petri dishes
You and me both!
So ive seen mixed reviews on lc and ive heard you dont want to carmalize the sugar. Ive tried tons of different methods and the lower sugar content method seems to be the best, and it seems to work with a clear solution
That was a very informative video! Great job!
I went on your website to see what liquid cultures you have available for purchase and they were all sold out. Do you have a timeframe for when they'll be back in stock? Thanks!
Hi Steve, we are working on having a better handle on liquid culture inventory. We are starting with a slow rollout but should have better inventory very soon. Keep an eye on the website over the next month or two!
If you had glass Petri dishes with a lid that allowed pressure to escape could you call your mixture into the dishes and pressure cook them that way eliminating the risk of contamination while pouring your agar
With the PDA recipe, how long does that last if kept in the fridge? Also, would you recommend adding Peptone and nutritional yeast to this recipe? If yes, how many grams? Thank you in advance 😊
Instead of plates, I use 50/$3.78 2oz Walmart condiment cups with lids. The lids seal so tight that I don't need to tape them. PGT even uses them in some of his videos. I fill them in a SAB and have a 90%+ success rate. When filling them stack them, and put one full of just hot water on the top of the stack to stop condensation.
Coool
Kool
High quality content subbed immediately 👌
Thank you!
Q: do you find any difference between filtering your dry powders for your liquid nutrient solution against not filtering ?
I personally always filter due to clumping or suspended particulates even when heated & stirred before PC'ing.
Great vid with solid info, Thanks
"Do you mean filtering the liquid after the powder is dissolved or sifting the dry ingredients to remove any clumps before adding them to water? Either step should be fine as long as you are adequately sterilizing your solution after filtering.
The particles and clumps are also fine to leave in your jar but may make spotting contamination more difficult. It is always a good idea to test your liquid culture on petri dishes or a small amount of grain before using it in a large inoculation project."
- North Spore Lab Manager
buy a stir plate or whisk better
a link to that pipetter would be awesome (please & thanks)
www.scilogex.com/scilogex-sci-fill-motorized-pipette-filler.html
What kind of yeast are you using?
Great video! How do you put the liquid culture into the syringe? Do you open the lid and extract it with a syringe, or do you need a long syringe needle to penetrate the self-healing extraction port of the jar lid? Opening the lid seems like it would introduce contaminates, but I am a beginner so I don’t know much about mushroom cultivation in general.
The second thing you said is the answer.
@@NorthSpore is this necessary? I can’t just grain to grain transfer indefinitely right? I’ve heard about how the mycelium gets “old” so I need to take spore samples and make into liquid culture or live samples? Also I’ve heard the original “patient 0” generation is better or more true than the future generations? Or am I wrong about that?
@@NorthSpore so to prevent “aging” it needs to complete its life cycle? I’m kinda new to mushroom growing and would love to hear your feed back 🙇♂️
@@NorthSpore and could I just liquid culture to liquid culture indefinitely? Or will it age like the grain to grain?
How to do Potato Dextrose Broth? is it the same method as agar?
I was kinda worried about having to grow mycelium on agar plates but if I can just make liquid culture and throw some spores in there with a syringe I’ll just do that. I have an 8 quart pressure cooker tho so I think I’ll have to reduce the volume and size of the jars I use
i use an 8 qt instant pot, i can fit 3 32oz/quart size like they showed. I think a fourth might physically fit in the pot, but interferes with the lid valves.
Are the lights above your hood regular fluorescent or are they some type of germicidal lamp?
Just regular bulbs!
Do you have a recommendation for a mixing/heating plate? I have not seen those before this video so I don't know what to look for.
It's a magnetic mixer. A metal pill is inserted into the jar.
@true source fire yeah but he said heat it .... do they have mixers that also heat it up.....?.... I have a magnetic mixer but it doesn't have a heat plate
Is instant yeast ok to use to make MYA? What kind of yeast really and if living, does it get thoroughly killed when autoclaving or some contamination issues can arise due to yeast being kinda heat resistant sometimes?
Yes, you can use instant and yes if you've truly sterilized, it is dead.
Great information and one of the most knowledgeable I've watched. Beautiful lab BTW.
Glad it was helpful!
Thanks!
Where did you get that shirt?!?
I would like to ask how to store both broth and slanted agar for long term use? do we incubate them on keep them in the cool environment?
Refrigerated will keep those things as long as possible.
Is PDA commonly used for storing cultures for long term/refrigerated?
Yes, it's very common for refrigeration, but not for a super long-term deep freeze situation.
Anyone know how to grow amanitas?
Does the liquid culture medium need to be sterilized in a pressure canner before inoculation?
100% yes
What is a Pie Pet?
Is it like a Domesticated Haggis with a Coat on?
So, for the MYA, what's the point of the yeast if you're going to just kill it off in the pressure cooker?
Nutrients, mainly proteins and fats
That is a beautiful hood. The lighting has a purpleish hue. Is there a uv component?
shhhh don't tell anyone... but for these lab skill videos we use an educational laboratory that we built in the office area of our facility. At the time of shooting we had this flow hood turned off to reduce sound. Lighting was actually grow lights placed under the diffusing screen which resulted in that pinkish hue. We use this lab for experimental projects and content production that doesn't require the level of cleanliness that our 'real' laboratory affords. We'll make a video soon to show you the real working North Spore laboratories which include an ISO 7 rated ultra-clean culture lab and multiple inoculation rooms. Stay tuned!
What is the tabletop heat sterilizer thing you're using for scalpel disinfection called?
Hello! It's a Bacti-Cinerator :)
Is there any particular reason to use Potato Extract and Dextrose for the Slants, and Malt and yeast for the plates? I've seen MA Slants before, but I'm curious if the PDA slants would be better for any reason.
Nope, they're interchangeable.
How can i tell if my inoculation of liquid culture is working? i used a 5% sugar to liquid rate with pleurotus columbinus and hericeum erinaceus (separately of course) but it has been 2 weeks now and i have not gotten a dense mycelial structure yet..?
The way to test your LC would be to put it on a plate or grain, see if it takes.
are slants usable for long term storage because they do not allow for air exchange with their closed lids? or would this lack of air suffocate the mycelium? I would like to store mycelium strains long term but havent had success with that
They have less airflow, more agar and less exposed surface area.
@@NorthSpore thanks for the reply. is it also ok to store genetics in the freeze?
I have a question regarding the recipe for the MY liquid culture. Are there any specific malts or yeasts that are part of the recipe, or will any type work? Also, do I need the hot plate, or can I stir the ingredients and place the solution in the pressure cooker?
I do not believe there are specific malts or yeasts that will and won't work. There might be slight variations.
@@NorthSpore Thank you for the clarification regarding malt and yeast. In a still airbox, could you clone a mushroom directly into liquid culture? Also, can you inject liquid culture into a plate or slant?
So 2cc for the LQ would be adequate?
Yes
When doing a transfer from an agar plate, you say the center area is less prone to contamination than the middle, isn't that the same area? Are you talking about the area between the center and the edge?
Yep, you're right.
can i make slants with light malt extract instead of dextroze?
Yes, you can!
13:02
Suddenly noticed something and found it a bit ironic.
All that sterilization and clean work but looking at it though a COVID lens all I could think was "But Where Is Your Mask!" Lol
I hate trying to translate mask muffled speech particularly on informational content
All in all pretty good info. Much appreciated. 👍
the room they’re in is a set they built- not their real lab! i had the same thought but they answered a comment saying it’s for filming content & other experiments
That is true.
Wouldn't keeping clean before PC simply be good practice?
I mean sure, but the PC takes care of things ideally.
Both my liquid culture and agar seem to have settlements, is there a way to reduce that besides filtering?
A little bit of sediment shouldn't be an issue, but you can try adjusting the cook time a bit or using different types of agar if it seems to be a problem.
I heard the term MEA once but I’m not quite shure how it’s made and what kind of use it has?
That's Malt Extract Agar, just another recipe, does the same thing.
@@NorthSpore thank you for the answer👍. Over the last month I also did further reading on this topic and learned that along the way. I am currently starting to cultivate mushrooms at home, also because of your videos🙏
I did the liquid culture and there is some white sediment at the bottom. Is that ok?
Ya, a little bit of sediment isn't a huge issue.
How is air gotten into the petri dishes ? Do they not need air to propagate ?
Yes they need air, but very little. A teeny tiny bit of air is in the dish and gets through on the edges even when you use parafilm.
Thanks for the great info! What are the lids called for the liquid culture jars and how are they made?
northspore.com/collections/lab-equipment
how long can u store them. like how many months max
It depends on a number of factors, but 3-6 months for plates is a good rule.
Hmm, sterilizing malt and water troubles me the least.
Sterilizing yeast will definitely kill the yeast, bad if you intend for bread or cake to be your feast.
But mycology is quite a different beast, so I'll assume we prefer our yeast to be deceased.
As far as the yeast goes, is not the nutritional value degraded by subjecting it to lethal, scalding temperatures and hyperbaric shock ?
Simply put, no.
Them desktop HEPA filters. Like Holmes. Pretty much hot glue a large clear sack onto the air output. Two holes for arms and a flap on top to load. It's sick
You don't need that crazy pipette. You can use a 10ml pipette which is way smaller.
Is PDA better for Slants, than say LME?
Nope, they both work well.
The alpha agar I can't ant find
He's like the son of Paul Stamets would be
I thought slants were used due to how tall they are so they can be stored in liquid nitrogen for long term storage of master cultures without the need to do culture transfers every 6 months or so like you must do when storing in a fridge vs freezing using liquid nitrogen
They are stored in deep freeze, but we don't have liquid nitrogen to my knowledge. It's just a badass freezer. Slants also minimize water loss.
What is potato extract? Is it starch or something?
Ya, pretty much.
He never showed how to prepare the culture media from the sterilized jar to the syringe inoculation step... We saw that he prepared the Malt-Yeast for the syringes, heat it, mix it and pressure cook it but, as soon as he pulled it out of the canner,(the MY amber clear mix, small jar) he had his syringe "ready" for inoculation... At what time did he showed how he 'got' or "input" the spores or mycelium or active 'culture' into the syringe? So you take the sterilized MY media and just inject it into a grain jar and SHAZAM!!! get mushrooms? (this never happened..!!) Well explained video right up to the "exclusion" of the "Empty syringe"... Gee
Bummer
@north spore can yall just write a start to finish encyclopedia already so i can pay for this knowledge. please and thanks
Working on it!
I'd trust you my life
in the PDA recipe you say to use 750ml water, but in the video it is clearly 500 ml in the bottle ??? is it correct recipe and recycled video with wrong amount in bottle ??
Go with the recipe, not the footage.
why dont you guys just put the plates right back into the Gamma ray sterilized bag they came in?
Oysters are not as good as morels, fine, anyone must admit that.
this video has too many points for one to follow. Start with an agar recipe and go from there. LC requires its own video.. Slants and plates are really similar except for the nutrient.
You can use either nutrient base for the slants or plates and it'll be ok. LC is pretty much the same except in liquid suspension instead of solid substrate
bingo
There will be many more videos!
why am i seeing terrance McKenna here?
Why on Earth ... rather Why In-LAB...
Would be have such a dirty looking Beard and long falling out Hair when working with these materials?
Also... did you SEE underneath his FingerNails... YECH!
That guy doesn't work in the lab much at all anymore, being one of the owners. Our current lab techs are both beardless. That guy Jon has a masters degree in Mycology btw.