Making Slants for Long Term Storage of Mycelium
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- Опубліковано 16 вер 2024
- Here is how we make our slants for storing cultures long term. We use Potato Dextrose Agar for our slants and a vertical laminar flow hood to do all of our culture work. Fresh from the Farm Fungi is a premium gourmet mushroom farm in Denver, Colorado for more information visit our website www.freshfromthefarmfungi.com and check out our social media @freshfromthefarmfungi
Lab Gear and other supplies: www.amazon.com...
That was very professional, informative, and enjoyable! Saw a few things I will definitely try! Thank you for the hard work and I definitely subscribed! 👍🏻🔥
great info but turn ur phone sideways bro you'll get more views
Plus, we want to see..& sometimes toss it to the tv or lab monitor..
Yea! What they said!!^^^^
Not here for the video quality.... but if that's what you want. there are people who know nothing and have nice videos
@@jameshorton7651 you misunderstand me, i obviously am here because i like the information but someone with less experience is attracted to the more polished looking content. We want good information to spread not just good looking information and for that to happen it must look good. I left this comment when FFTFF was a much smaller channel and he has sense drastically increased his video quality and has become one of the largest and most respected fungi channels on youtube because of it.
Great video but it’s weird to watch it in the portrait mode.
Very weird. Someone held there phone the incorrect way.
this was back in my early days of filming
Thanks so much for all your videos. Much needed info in one place. It’s nice to have someone with a technical background breaking things down. I
Great work Gary! Your videos have just the right blend of quality information. Much appreciated.
Sir! You are a valuable source of information. Great instructions. Mush love.
Shared your video to MushroomMediaOnline and MushroomGrowingBusiness on Facebook keep sharing helpful tips.
Thanks Gary for your time. Very informative.
Thanks for the info! Question. Can you elaborate on “It’s better to inoculate right away and use parafilm around the edges”23:35? Parafilm over the cap or replace cap with parafilm? Sounded like a useful tip but I’m not too familiar with that procedure or technique. Just another aspiring rookie in the Myco world. Thanks Gary.
a-gar vs ar-ger one of them is an old fashioned oil-burning stove
I kept wondering what the hell was « auger » XD I was like « the thing to dig holes??
@@oOVanillaMelOo
They do sound very similar, especially on camera talking over a flow hood.
Great video! I like how large the tubes are that you used.
P.S.
Be careful using bleach and alcohol. When the two mix, they produce chloroform!
2-chloro propane
50ml
Thanks for this informative video. Keep em coming please 🙏
🍄♥️
Gary thanks very much,great video,great infos.Dieu vous protège.
Excellent video, thank you.
wow! really great learning Gary, ty!
Thanks for using metrics for us non states civility
Great instructional! Mush love!
Cat appears in the background at 7:28 =) You're welcome!
I found the last 2-3 minutes the most useful. I was looking for information on how long they keep and how long a strand could viably sit on them. I was hoping for more information about the freezing option. This video had lots of nice tips and a cat!
same. It seems harder to find the correct info on how long agar media and cultured agar media will last if stored properly.
@@rexcracin8642 I did see on FreshCap's website, he said he has lion's mane culture sitting in the fridge for several years and still works fine to start some spawn.
Great stuff. 🤘
how about put agar in slants, than put 4 slants in a mason jar and PC them... than let them cool down and bingo... and if u need storage for up to a year a agar plate or agar dish will do the job.. they last well over 1 year !!! just seal them up...
A small amount of mineral oil on top will prevent the drying out and can be stored longer
I use a 2-10ml pippette and 15ml tubes.
I only have a refrigerator with a temperature of six degrees, is it possible to store in it?
and you usually transfer all your slants every year? how many times can you do that before it is unusable due to senescence/mutations?
is it better to start over from spores every 1-2years?
thanks for your content, it really helps
doing this really slows down the degradation so it preserves the strain much longer. It depends on the individual strain but they can stay viable for decades this way - starting from spores is a good way to find better strains so I recommend continuously breeding to improve the overall performance of the operation but slants can help preserve them for a long time
So you can always go back to these stored agar cultured slant to keep making G1 spawn ??
I appreciate the clear and precise description of all your doing. My question is in regards to the slant tubes. I’ve seen folks putting a toothpick in with them? Do you do that? Why or why not? If so, when and how do we incorporate that?
It’s just personal preference - the idea of adding tooth picks or popsicle sticks in the media is to add more nutrients for long term but I have used slants both ways and don’t notice a difference -it may be helpful with more finicky strains
Wow that was worth watching
To store indefinitely with glycerol and freeze, Do you add it to the mix before pressure cooking? 2nd question Will freezing destroy spores if you wanted to store spore syringes indefinitely?
to store "indefinitely" with glycerol you need to add sterilized glycerol to the surface of the mycelium and completely cover prior to cryo freezing (-80C or below) the glycerol will provide a protective sheath that displaces the water inside the cells and prevents cells from lysing. Spore prints will last much longer then hydrated spores but refrigerating syringes of hydrated spores will last for a very long time (years). Freezing them as-is will kill them or reduce their viability significantly. Another option would be to add glycerol to solution (either liquid broth culture or spore solution) and freeze it this way. There is more risk using liquid for long term storage as when the mycelium thaws, it is very fragile.
How many slants do you do for each culture you want to keep long term?
Hey Gary, love ya channel and how you present the content. Can I ask you a question mate? In this video you mentioned that you prefer potato dextrose for long term storage in your slants. My question is, Why do you line to you potato dextrose over another? Cheers mate.✌️👍
I also use MEA and V9 for long term I just like to rotate them through and PDA holds up well over time. No real reason ha just biased from my own experience but always open to trying new ones this is just the best I have found so far. Hope that answers your question! 🍄❤️
Hello sir
I want to ask about one cans have potato dextrose and agar?
Dose that shorten the time a culture will last if I use a 15ml?
I think it might but I haven’t tested this so it might be negligible. The reason I use 50mL is because they are easier to dig into with a scalpel
Thanks for the info buddy! I just started making my own ogg-err too. Ogre is the best media to store maycelium... I also love to grow Mooshrooms... 😂
Love using Auger.. also Layquide calture is good too!
Good ol auger, it's the best for mycelium cultures ;)
I've seen other videos about slants where they put pieces of wood, like popsicle sticks, inside the tubes. I'm not exactly sure what the purpose of this is. Have you done this in the past?
So it is seal... But the mycelium needs to breathe right?... Don't you need like a filter to let air pass?
Hi there, if LC is good for at least 8 months and slants for one year I dont understand why its called long term storage ? only if you can freeze it really. How can you preserve long term your genetics as some mycelium should never get under 55 degres as it would died ? Do you agreed that perhaps the best way to preserve genetics long term (years) would be to keep your collections as spores prints ?
Spores are the best for true longterm storage. But that is a randomization of genetics. I think the term “long term storage” is relative. As in relative to keeping Petri dishes out at room temperature. Won’t take long for Petri dishes to become overgrown. This is a good way to keep mycelium for a while.
I'm hooked. I want to learn everything I can about mycology so I can make medicine and replace meat.
@@s7r49 I like meat. I'm a pit boss..but if the killer rabbit virus, or the squirrel bubonic plague, or pig covid decides to infect and kill everything, I'd like to know I'm eating well and really don't need meat. Next, there will be a zombie cow disease, and killer chicken flu that eats antibiotics and vaccines for lunch,and a little more pollution if not parasites because of the water temps, just like in an aquarium...and meats will be inedible. So, smoked oyster mushrooms, chicken and lobster mushrooms, shitake I know how to grill..and if I blindfold you, you will swear and bet you're eating the best prime rib of your life...that's what I'm talking about. Gourmet grow tents, here I come. And a solar freezer. Maybe a small cryo..not to mention the meds..antibiotics, anti inflammatories, muscle relaxers, migraine, anti depressants, immune boosters, it's all in mycology and horticulture.
I haven't seen isopropyl alcohol in 3 months. We've been having to pick up prescriptions 4 or 5 pills per trip. We live too far away for that, & it's just going to get worse..all our meds come from China.
No im hooked.... I wanna get high 🤣
Hey man cool vid thanks.
Why don't u use honey as antibiotics?
It's good to freeze Aswell right?
Why no wood in the slants? My understanding is that extends the storage life considerably even if the species isn't a wood-lover
I have used both ways and don’t see a difference but it could help yes
Ya good stuff. However, Slants are "Outdated and Old-school".. Want long term storage without refrigeration? PC some tap water in a tube/jar/container etc. (yes, tap water, you can use distilled but you don't need it. the minerals are beneficial) for an hour at 15 psi. Take a chunk of your agar plate with your strain you wish to store and put it in the sterile water once cooled. (can put as much in as you want to store, but don't over pack it)
Put a cap/lid/stopper on tight, and that's it... Done.
Op. seal up the cap with cling wrap, parafilm, or grafting tape for extra protection if you want.
It will stay good for 4-5+ years as is.. Slants are Out dated. I use 20 mill cryogenic tubes and have my entire library of 120 strains in a small container slightly larger than a big mac container, sitting on my lab bench.. I've come back six years later to one I forgot I had and it was just as fresh as the day I put it in there. Hope this helps anyone reading this.
this is rubbish. but thanks for the algorithm boost. I can say yes, LC’s can last years or even decades but the reason for slants is that you can instantly see if it’s healthy and clean. There can be contams lurking in liquids and it’s not a good gold standard to rely on.
@@FreshfromtheFarmFungi .. it's garbage because you say it's Garbage? I know why you make slants. I was not asking you a question like this is something new to me. I've Been at this along time young man. Yes, slants still have there place and I use this still also when I'm working on a strain for long periods of time. I'm not being rude about your video, I like your work. I'm just bringing up another method I use.. take it or leave it, but rubbish it is not.. this is not something I made up, it's actually a tech my dude..
@@FreshfromtheFarmFungi I think you may have mis understood the instructions. You would have already established its Health and cleanliness because the culture has already been grown out on agar. Your taking a chunk of that culture (agar and all, like you would use to inoculate your grain) and placing it in PC'ed water in a jar or tube and suspending it in the liquid . It does not Grow in the solution like a slant may, but that's the point. its just remains dormant. Its for storage not long term use like a slant.. I legitimately am not trolling. I'm just giving you a tool for the bag is all.. I don't care if you use it or not. 😁
@@FreshfromtheFarmFungi .. very confused with your response. you said "Its Rubbish" then turned around and said yes it last for decades.. so is it rubbish or does it work for long term storage. Thats not very nice. especially since I was only making a comment that you (after calling it garbage) said works.
So by "long term" we mean about a year?
We transfer from slant to slant every 6 months or so but they can survive many years depending on the strain
Love these videos! One question though.. How specifically do you store the slants in the fridge? Do you screw on the caps fully post inoculation so there is no air exchange in/out of the slant?
ua-cam.com/video/FTbHO2ql8X0/v-deo.html watch this one there is more info about that
How do I start working in the hood? ;D
How long do we need to wait for the agar slants to cools down and solidify? I prepare half-strength PDA slants and it takes more than 3 hours to solidify
Good Video
mush love Gary, you just helped me figure out where my contamination is coming from (letting the PC cool on the stove)
looks like I am building a fume hood !!
do you by chance have a links with prices to some good filters ?
Thanks for watching! Check out air science they are my go to! Great customer service just ask them what you’re looking for - MUSHLOVE
Better be carefull working in the hood I hear it can be dangerous.
But can we grow mycelium from slant sample? Say one had not access to spore but fresh flesh??
ipriori Yes tissue samples will grow on slants as well, the focus here is for long term storage though. If you use tissue it is much more likely to contain contamination so the best means to take tissue cultures is on petri dishes and then take the clean mycelium and store it in slants - I will make a video explaining the tissue culture process! Thanks for watching and MUSHLOVE
Why not just autoclave the slant tubes with agar already in them?
you can do that as well in my experience they tend to get more watery
I see you have a panel at the top and sides of your flow hood. Obviously that’s to direct the airflow towards your self. Is this a necessary addition? I don’t have that I just have a regular horizontal laminar flow hood with a shelf in front of it
check out our video on the differences between vertical and laminar flow ua-cam.com/video/v5l5yNCxsBs/v-deo.html
So i can put the slants straight in the fridge after inoculation?
no, wait for it to grow out a bit first it helps with recovery
Thanks for all the info! I read that thing about the pressure cooker pulling in contam in Stemets book and I don’t understand. It’s got positive pressure and it’s really hot so wouldn’t air/energy always be moving away from the pressure valves? I’ll get a hood one day. Great channel. Thanks again
When it cools off, it sucks air back in. It’s easy to observe if you have a pressure canner with a weighted regulator.
@@covid-19ispsychologicalwar10 thanks for the reply. I guess I don’t doubt that it happens, I just don’t understand why. Maybe fluid dynamics isn’t something to be learned from the UA-cam comments section?
@@zachkiss8870 Basically, your cooker is filled with normal atmosphere when it's cold. You put it on the stove, and it starts generating steam. Lots of steam. The regular air inside of it is pushed out by the steam and by the time you're at 15 PSI, it's all gone. Now it's just steam. As it cools, the steam condenses into liquid, leaving behind near-vacuum. If your cooker doesn't create a vacuum, it'll suck regular air back inside to replace the vacuum. That's why Stamets says to put it in front of a flow hood while it depressurizes.
Subscribed! :) But I have a question. You said slant. I thought slants were the little glass things that go under a microscope for viewing? Is a slant a tube like you showed?
Jeffrey MOORMAN Thanks! Slides are little glass surfaces to put things on for microscopy - slants are tubes of agar like in the video - sorry for any confusion MUSHLOVE!
If one is reasonably sanitary, how likely is it for a slant to get contaminated if one takes some samples off a slant and puts it back in storage? Is it not recommended to do so?
It can be done easily since the slant has a narrow opening, just make sure the tool is sterile and the airflow is sterile and the cap doesn’t get contaminated 👍
Hi, Thanks for your video. May I know what is the temp setting that you use for your Petri dish incubator?
73-74F
may I ask why you wouldn't put the agar in the slant tubes and then sterilize them?
you can do it that way but these are plastic
Ahhh. I just bought some plastic ones that were similar that said they were autoclaveable. I’m going to give it a go. Thanks for the great videos and for the etsy page. I ordered some tortoise shell beech culture the other day.
awesome! It may work I just have had them melt in the past and do it this way - eventually Ill invest in glass tubes and metal trays but there are other priorities 👍🍄❤️ goodluck with your project 🙂
Are you spraying the surface of your laminar flow hood? I always clean the outside of the hood as well as the back section as that’s where the air enters from
We clean the whole hood, change pre-filters regularly and clean the work surface before using every time
Wouldn't resealing the bag over the slants not provide the mycelium with oxygen?
There is enough headspace in the slant tubes for the mycelium especially going into the refrigerator which slows down their metabolic processes
What temp in farenheit are you pouring these? I normally pour agar plates at 120-115° farenheit. I feel like I could pour slants as soon has my gloves will allow me to around 150° so they don't co so I can't set them as a slant angle
I like to start them around 130F that is the sweet spot
Hey Gary thank you very much for all your knowledge and videos I do have a question in some of my syringe inoculated perti dishes I have different stuff growing other than mycelium LOL not funny🙃 no flowood unfortunately at this time but I do have strong mycelium growth and I don't want to lose my genetics can I take a piece of healthy mycelium from an agar petri dish that has foreign growth and transfer it to another . Thank you Gary I appreciate you
yes transfer from the clean outer edge of the mycelium it may take a few tries to get it clean but it can be saved
"I can wipe from back to front" Me too bro me too but I keep getting yeast infections in my vagina.
hah this might be a useful joke someday thanks
@@FreshfromtheFarmFungi 😂
How do you store your lab coat and where in your lab? Also, do you wash it after each use?
I have multiple lab coats and hang them up at the entrance so I can use a fresh one each day or so and then wash them as needed
Wanna party iam a fungi classic 😂
This probably a noob question but how does the mycelium get oxygen in the slants when there is no ge hole ? Would the c02 build up to the point it would starve out myc ?
there is some small air exchange through the threading while it incubates but once it colonizes I apply parafilm and put into cold storage where respiration decreases significantly and the mycelium goes dormant. Think of all the mycelium underground during winter.
@@FreshfromtheFarmFungi dude you are the best !!! Thank you for replying dude but just one more thing I’m about to start my first slant I was just wondering after a year or so how do I maintain the culture to make sure it’s still viable in cold storage ? And what temp is good for cold storage you the best dude !!
If you have filters or leave your lids tight you wouldn’t have to worry about contaminants getting into cooker while depressurizing 🤙🏼
Indeed! thanks for the tip! and thanks for following along MUSHLOVE
Fresh from the Farm Fungi LLC and if you don’t mind me asking why not just pour your slants before pressure cooking?
@@tracehormann6188 That is a great question - so I have done both ways in the past and it is definitely a standard way to make slants, (we used to do hundreds of slants for plant tissue culture a day in one of the labs I worked at) However, I prefer to pour after autoclaving because of a couple reasons: The main reason is that I usually do slants and plates in the same batch so it's easier to mix a large batch and pour out what I need accordingly - Also, the caps on slants are "loose", so when you autoclave with media in them, there is a displacement of water that can't be avoided - it's not a huge deal - but often you can end up with "watery" slants on the bottom and super dense slants near the top rack and I like the homogenous end product of pouring them all afterwords. Like many things in mycology - it comes down to preference you should do whatever works best for you. MUSHLOVE
Fresh from the Farm Fungi LLC that’s interesting. I’m about to do my first slants and appreciate the info!
I understand pink oyster dies on agar if put in the fridge?
Not true - I store mine at 34F and it’s still living. Possibly is strain specific?
Whats the biggest difference between slants and plates besides what its put in? Why does it last long term?
Slants have “butts” which is a deep well of nutrients - if you look how they are poured the media will last significantly longer. Plates are very thin in comparison and will dry out much faster - hope that makes sense
@@FreshfromtheFarmFungi makes perfect sense, thank you I appreciate the reply. Have a good rest of your day!
this slant's work for cordycep culture, if yes where we put this slants in ice chamber in fridge or at out of ice chamber generally tem range is 4-5° c
Digvijay Chauhan Yes it works for cordyceps, keep it in a dark fridge 34-37 degrees F and minimize exposure to light when making transfers to prolong storage since cordyceps senesce relatively quickly compared to other mycelium
S7R4 3 months should be fine - from my experience they degrade after a year or so or if they are transferred multiple times in sequence (going from plate to plate or plate to lc back to plate) etc. I like to back up G1 on slants and they will be good for years
Can these microfuge tubes by pressure cooked?
yes, not sure about the stand though
@@FreshfromtheFarmFungi Thanks!
7:55 someone is hungry!!! Mrrrrrrrrrr
Cant you just make a small jar of inoculated jar of grain spawn and put it in the fridge for long term storage?
for up to a couple months yes - this is for 6+ months of storage
@@FreshfromtheFarmFungi I see, thank you
jump scare at 19:45
What is the difference between storing in petri dishes or slants if the medium is the same??
I will make a video about this soon, there isn’t much of a difference but it’s enough to make it worth having both.
@@FreshfromtheFarmFungi thank you
alcohol doesn't do anything for endospores or yeasts, 7-12% h2o2 has been shown to have sporicidal effects
Awesome thanks for the feedback! We clean our hood with 15% bleach and wipe clean with alcohol - Air science also makes hoods with built in UV lights as well and you can implement Ozone generators for spore mitigation as well : ) MUSHLOVE
Never wipe back to front bro! Ew! Non sterile practice right there. Lol that being said I enjoyed the video
what type of centrifuge Tubes are you using ? are they DNase/RNase free?
yes and sterile
@@FreshfromtheFarmFungi , how can you keep the tube to remain DNase/RNase free after pouring the agar?
I believe that DNase/RNase contaminants will not be killed even with autoclaving the agar.
thanks! Can i freeze it for long term?
There are methods to freeze for very long term storage but it requires a flash freezer and glycerol so that the water inside the cells are displaced until thawing. I may make a video in the future on this subject
With the dowel, craft stick, whatever, they will last quite a long time in the fridge. I soak my pins in a water/dextrose/yeast solution. I've had cultures still alive nearly 2 years later using a 1/2"x1 1/2" dowel pin. Just keep them inside a sealed clean container; even a clean fridge isn't exactly a sterile environment.
My god
Awger
Okay have I been saying agar (Ay-gar) wrong this whole time🤦🏽♀️
It’s the western NY/Buffalo accent a lot of people say Ay-gar but I can’t unlearn what I was taught hah
why are you wearing headphones while filming. You've mastered mycology but not basic camera skills.
Don't use bleach and alcohol together
I spray with bleach, let it dry, and then spray with alcohol and wipe clean - I agree don’t mix the two solutions together though
@@FreshfromtheFarmFungi drying isn't going to be sufficient enough to ensure there is no chlorine content. Spraying them in the same work area is just as good as mixing the solutions. Take this video down before you kill someone.
I hate bad titles... you did not make slants. You USED slants.
Ager or agar?
Say agar properly
I do say it correctly - there are many pronunciations Im from western NY and was taught in University to pronounce it this way
Great video but sorry no like I cant do hit 666 already got enough bad luck 😬
Auger?? 🙄🙄
It’s pronounced agar not auger
ua-cam.com/video/IqpCJV0OPd0/v-deo.html
packardcommunications.com/2018/10/30/mispronounced-words-american-english/
I'm willing to bet you do a actually say some of the words in that link incorrectly. I would like to say in my years of learning mycology it is almost always pronounced as "Aye-gar".
Further more here is a rebuttal to that UA-cam link. BTW it's from Cambridge Dictionary.
dictionary.cambridge.org/us/pronunciation/english/agar
dictionary.cambridge.org/us/pronunciation/english/agar
Haters gonna h8. I'm from Texas and I'll bet 90% of my pronunciations would drive 90% of the world crazy. I pronounce AGAR like a pirate with a extra helping of RRRRRR and hope no one goes nuts over that. I live in Tennessee now because everyone here speaks normal. (LOL)
I can't believe all the time being spent on arguing about pronunciation when we just received some top notch information from todays video lesson. Mush thanks
Pronounced A-gaR not ah-ger,, lol
Its Agar not augar
Gary Rose Throughout all of my academia and years working in clinical/microbiological labs this is how it was pronounced. I’m from upstate NY - it may be a regional thing but I do indeed mean the same thing. MUSHLOVE
Stupid vertical filming
What if I don’t have a hood 😢
you can use a still air box ua-cam.com/video/X1GjK7vJVpI/v-deo.html
@@FreshfromtheFarmFungi thank you much ❤!