Inoculating with liquid cultures and utilizing media extraction techniques for mycology
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- เผยแพร่เมื่อ 25 มิ.ย. 2024
- This video is about Inoculating liquid cultures and utilizing media extraction techniques - The media extraction device was produced by Lion's Mane in Denver, CO. Check out their website for more information lionsmanedenver.com For more information about our business visit freshfromthefarmfungi.com and to purchase our cultures visit our ETSY shop FRESH FUNGI www.etsy.com/shop/freshfungi MUSHLOVE
Gary's Recommended Products: www.amazon.com/shop/influence... - วิทยาศาสตร์และเทคโนโลยี
Wow, nicely done. Very informative and helpful. Thank you for making them.
Thanks for the info and your time!!!🙏🏻
Really informative video I learned some key things that I will be doing from now on with my LC it's very easy to pick up what you are explaining I like how detailed your videos are. Will be hanging out watching for the next one after I subscribe. Thanks Gary
thank you for this video
loving yr show and info ... keep it up ...learning so much from Australia
Very nice, thank you.
tanks for the vids man! you inspired me to make a lab and start a mushroom farm!! mush love fam
i appreciate you making the distinction between aseptic and sterile, huge pet peeve of mine
Gary, I really appreciate the QC / Test portion of this video - I need a lot more of this sort of thing for our process. We're learning the hard way as we go and your videos are super helpful - thank you! Where are you getting your QC plates and other lab supplies if you don't mind sharing? Thanks again! George in Idaho
Neo Geo Thanks for watching we appreciate you’re support! We get our QC plates through Hardy Diagnostics and our lab supplies we source all over depending on our needs : ) Hardy Diagnostics and Thermoscientific are good brands
Greetings Gary, your videos & the information you provide are some of the very best. Thank you for your commitment to excellence and your willingness to share some of your hard-won wisdom with the rest of us! I wanted to ask you about your blood agar and the hektoen enteric agar plates you utilized in this video. Do you make your own, or do you purchase those plates pre-made from a supplier? I would sure love to see you do a comprehensive video on quality control strategies... thanks in advance for your response, Merry Christmas to all of you out there in Colorado.
I started straining the lme in a coffee filter from the water after it’s all mixed up. Then pc it. Crystal clear.
I bought some Turkey Tail from you, amazed how fast it grew on agar plates! Highly recommend this grower guys.
Thanks for following along! MUSHLOVE
awesome. im excited for you LOL go for it!!!
YOU ARE AWESOME !
Hey Gary! Once again, another super video. I am all about QC, that being said, would you share which of the blood agar plates you purchase from Hardy? There are a lot of them. Also the one you use specifically for salmonella and coli?
It is useful video, . Thank you
Hey Gary I know this is an old video, super appreciate your video been following along for years. My question would be do you have any advice for using these media extraction lids in a SAB environment? Especially as far as keeping the luer lock sterile
Safe to say.....really excited.
Hi Gary! Thanks for another awesome video, thanks for sharing! I couldn't help but notice the cracks in the walls of your lovely laminar flow hood. Is there an interesting story behind that, perhaps one that involves a pressurized vessel unexpectedly coming apart?
Brilliant
I use a three way valve in some ozone therapy. It's probably not the best solution, but it would be straightforward to have a line in to the valve, and the other two spots connected to the syringe and the needle, in a linear fashion.
Vert nice I would just use the LC jar and pore it in to my grain but would be nice if you make a lot of LC syringe to make.
Dude, any luck with the morels?! Can't wait to try and clone some next spring!
I like the new setup have done lab professional hope morels take thats 👍👍👍 thanks for the video
I'll be doing this experiment shortly.
I want to build a few of the media extractors. Having trouble locating parts though
I work with almost the same setup, but I insert an sterilized automatic syringue in the big jar through a silicon tube and a needle. The grain jars have an injection port, so I can inoculate hundreds of jars in minutes without opening them, saving time and possible contamination. Also, I seal the bags before sterilization, and do the same as before through an injection port.
石头 awesome that sounds like a great and efficient setup
@@FreshfromtheFarmFungi Also, I'm working with that setup to inoculate logs in the forest in a most sterile and fast way than the traditional, with only a battery drill and a peen hammer. With the help of two friends, one drills narrow but deep holes in the log or tree, another insert the syringue and inoculats it, and the third one only needs to close the hole with the peen hammer, like when you make a rivet. This way, there is no need to drill big holes and fill them with spawn, or using wax or stoppers to seal it. But I'm still not sure about it's viability.
石头 would love to see some of the results! MUSHLOVE
can u post some videos
Love mushrooms!
Fantastic my dude. I was wondering if you were going to do anything with morels. I just knew I had to be patient.
He's still trying to figure it all out if you haven't kept up
@@blacksheepnomad9000 Iade that comment a year ago dude
@@kodyfrost and here I am and here you are and he's still trying to grow morel's. time is unimportant. what we do with it is. For instance I'm keeping up with his efforts and your salty I updated you
Nice Hermano Mushlove 🍄
Are you active on shroomery or any other forum more suitable for asking questions than YT chat?
Hermano me has descrestado con tu herramienta, solo x esto mereces mi suscripción y aca estaré pendiente de tu contenido
Hey! Love this video, the media extractor, and your technique. Are you using a vertical hood? Are there specific qualities I should look for in a honey? What size test tubes do you use for your slants? What type of tissue culture/solution was in the vial you used to inoculate the honey LC and how big was it?
instablaster.
Amazing bro good jo.
Love the videos Gary! Thank you 🙏
17:05 just loving it
Hi gary what type of tubing did you use for your LC? I heard some type are permeable.
This reminds me of my work in the OR. Wouldnt hurt to wipe the tube tip before placing the syringe with alcohol wipe to avoid any contaminates on the tip of the tube.
yes similar aseptic techniques - I agree that could be a great extra precaution
Well done. Really interested in the pnematic pump idea
When you come back after "a few days", was the media being mixed the whole time, or just when you put it on for the videos?
I thought at the 24 min mark when you moved the extractor from one jar to the other jar.
You did not sterilize the extractor.
Would that contaminate the culture ?
Or did I miss something
Hey Gary 👋 I gotta get me a media extractor!! Thing is awesome man😎 Does the hose unclip from the jar lid to use on multiple LC Jars or do you have to move the lid and all to another LC container. Cleaning it out in between of course 😉
Hey thanks for watching! They are all one piece so I use a normal lid for storage and have the extractor lids on hand in a sterilization pouch - reach out to Matt at Lions Mane though he would appreciate some feedback and can make custom orders if it’s in his wheelhouse
@@FreshfromtheFarmFungi Yeah, I was just watching this and wondering if there is any reason I couldn't make my own DIY version of this lid that would fit on a mason jar. 🤔
Please tell me what’s the name of that first gel and the second one? Liquid culture process
Great job! I understand so much about lab thanks to you. Can you describe your tools sterelization process? Thanks again from France
Thanks for watching! We sterilize everything at 15PSI/250F for 20 min (liquid) 60 min (tools) 90 min (substrates) depending on batch sizes and the sterilization pouches or strips are great for confirming sterilization
@@FreshfromtheFarmFungi HELLO THERE 🙂!
WAIT SO IS THE HEAT OK FOR MY LC WHILE I'M STERILIZING THE WAY I BELIEVE YOU DID? I DIDNT UNDERSTAND QUITE WELL AND I DON'T WANT TO KILL THE LC THAT I RECENTLY ORDERED? I AM GOING TO INNOCULATE MY GLASS JAR FILLED WITH HONEY AND DEXTROSE (MIXED ALONE) BUT I WAS UNDER THE IMPRESSION THAT YOU SHOULDN'T ALLOW LC TO GET ABOVE 70 DEGREES OR IT DIES. IS THAT CORRECT? EVENTUALLY, I PLAN TO SPORE THE LIQUID CULTURE ON GRAIN AND MANURE SUBSTRATE.
SORRY FOR THE CAPS I AM NOT SCREAMING JUST DYSLEXIC AND A BIT BLIND🤓😝. I AM NEW TO THIS FIELD AND I AM DILIGENTLY RESEARCHING AS MANY GROWERS AND THEIR PROCESSES AS I POSSIBLY CAN.
IN YOUR OPINION, WHAT STRAIN DO YOU RECOMMEND IS THE EASIEST AND FASTEST TO FRUIT IN THE MAGICAL MEDICINAL AREA? GROWING TO LEAVE BEHIND THE POISONOUS BIG PHARMA MEDICINE TRAIN TO TREAT MY PTSD, CHRONIC PAIN AND DEPRESSION NATURALLY BY MICRODOSING.
ANY ADVICE, DIRECTION OR HELP FROM ANYONE IS ALWAYS APPRECIATED. LONG LIVE THE FUNGI WORLD! 💚✌🍄
How much rice per Lc do you use? Great content btw thank you!
Hi, so you don't use Peptone any more or don't think you need it...interested to know...thanks!
dude, thanks so much man. such a great video, no one has made such a in-depth tutorial about liquid culture. why am i not using that extraction technique!!
idk if you'll read this but can i ask what is your job title?
Hey thanks for following along! I am a mushroom farmer, scientist and entrepreneur - professionally, a Medical Laboratory Scientist but have found a passion for growing food and mushrooms are my gift I embrace MUSHLOVE
What’s the first gel & second gel please let me know
Thank you!
That's nice I'm interested in getting morels going we had drought and there aren't any more it's a shame. Tried getting some established no go 😞
I use my own maple syrup that I make and water. The way I figure It, it comes from a hardwood maple tree. Tress are what mushrooms love. So it contain s the same carbohydrates it’s used to.
idk how true that is but i love teh logic!
Any luck on the morel growing ?
Hi Gary. Nice video. I like watching your videos. Please at what intervals do you mix your LC on the mixer. And how long? Sorry for translation. I'm sending you greeting from Slovakia. I wish everyone well.
thanks for watching! I usually mix once a day for a minute or 2 at the least and sometimes longer to break up larger growth. It can keep mixing as long as it is sterile it won’t harm the mycelium. MUSHLOVE
@@FreshfromtheFarmFungi how long could one keep healthy mycelium in a refrigerator, assuming it passes qc?
@@AspireBlack It really depends on the strains and genetics of the mycelium being stored - I prefer to store genetics on slants long term. But in LC cordyceps will last the shortest before degrading and some species like reishi can last for years.
I'm amazed by the informative and quality content that you produce. I'm an aspiring hobby mycologist but I have plans of perhaps one day making it official. I've absolutely fallen in love with mycology. I'm starting from practically nothing but I have full intentions of one day having my own mycological business and lab.
:)
Mush love
thanks for watching and following along🍄❤️
Nice and informative videos
I do have a question fresh from the farm fungi how long can you keep using the same culture before it’s not good
it depends on each strain - we store them in slant culture tubes to preserve the mother culture for years and years but some will last decades
Can you tell me what you need to make mushroom juice?
Great video Gary! Curious how you got all those cracks in the acrylic windows of your flow hood though. I just bought an AirScience Flow like yours and I’d like to avoid that happening!!😮 Keep up the good work man!
It happened from setting bulk substrate bags against the sides it caused it to heat up and cool down which made the cracks. I replaced them fairly cheap (100$ from the hardware store) but now I only use this hood for culture work and have a horizontal ffu type unit for my bulk substrate inoculations. I love the air science hood it was just a user error on my part haha
@@FreshfromtheFarmFungi Gotcha, thanks for the heads up!! I'll keep any big heat sources off the panels.
I would love to hear a follow-up review or comment! Are you still using the media extractor? Also, after the jar is done, is it easy to sanitize and clean the tubing?
yes I use these still Im making another video with a new ad-on but they are easy to clean I replaced the hose about twice a year because it gets loosened
What type of agar would you recommend for morel long-term storage? I guess also for other mushrooms as well if the answer would differ
Check out our video on making slants for long term storage - we prefer MEA and PDA because many strains do well with it! You can also lean a toothpick or popsicle stick inside the slant and sterilize to help with even longer storage. MUSHLOVE
What size pressure cooker do you use?
Referencing to when he says use just honey : Use whatever broth for the LC you want and whatever works best for you, you're not gonna be able to see contamination anyways and you should always be running agar before you noc anything
In the video did you transfer the media extractor from the fresh mix of cordiceps straight to the morchella? (@ 24 min) cross contaminants?
It was sterilized between
is the air exchange of the spawn jars from lids upside down and loosely screwing the bands on?
yes it doesn't require much at this stage
I’ve spread many mushrooms while dropping spores placed them A blender with molasses water mix and store them in jugs shaken every day and spread around my yard . Mushrooms galore . I’ve done this with amanita s as well
awesome! I always wanted to try this with a backpack sprayer 🍄❤️
@@FreshfromtheFarmFungi that’s a good idea. I think the reason molasses works well for me is the fact that mushrooms love sulfur and some sugars . Now I’m going to try your method. I’ve never tried honey but it makes sense honey is anti bacterial . I wonder if when I hydrate the honey more, will it lessen the anti bacterial strength. My bees take a long time getting the honey to the exact hydration so it won’t mold . This is very interesting maybe I’ll add a touch of organic sulfur.
Thank you for the video. How do you make blood agar plates and green plates? What is there except for agar and water? Thank you.
th-cam.com/video/iHYBu3rXHes/w-d-xo.html
what do you call those hose that you connect to the top of your reagent bottle ? Im looking for those to use with mine thanks :)
Flex tubing, pharmed tubing, silocon tubing it is common in most micro labs
could you make a video on how to make those lids for the media bottles?
matt from lions mane equipment and supply in denver makes them if you reach out he might be able to help 👍🙂
This was great. What would be cool is if you attached a third tube to the bottle with a needle on the end and just used the syringe side to draw up and pump out with either a tap or valve to force the liquid outbound. I did see the selection and breeding videos, so awesome. Have you made anything on microscopy for isolation or breeding?
Cool suggestions Ill pass it on to Lion’s Mane equipment supply store 👍 Check out the series on Cordyceps - we did a microscopic ascospore isolation in the first video - I prefer to use dilutions because of the process it’s less time sensitive but both will work
If you forced air in to push out liquido culture you would basically be introducing contaminated air into the sterile atmosphere within the jar.
It defeats the purpose of the jar sterilization process.
@@Lalovision3D obviously you would not use air that was not filtered.
What about an extra long needled syringe that can be reused and re-sterilyzed. Does that exist? I will have to look..
Great demo of the extractor. Tried to order one but they are out of stock "bummer" Also can you show how you make the blood agar for your QC plates?
Thanks for watching! I think they take pre-orders on their site : ) Also, we purchase our QC plates from hardy diagnostics (that way they are third party tested and certified) but they are made the same way as any other media - MUSHLOVE
I finally had success with inoculation of Morchella around my new house. I found one variable that allowed almost 100% success. All the other spots I tried did not fruit. There’s something that everyone sees every time they hunt Morels but they never take note of it. That’s my secret. 3 years straight with success.
It’s not really how you make your spore slurry or grow your mycelium but rather, it’s where you put it that matters!
cool! when did you spawn? Also, what species?
@@FreshfromtheFarmFungi October. no spawn. Just spores from nothing special except dried morels from spring. Distilled water reconstituted and then 48 hrs with molasses. Then straight into some slippery elm leaf mulch. Then placed in *specific areas*
No offense to your scientific method but nature doesn’t allow for complete isolation from mirobrobes and does just fine. I’m just a simple man with a simple plan :)
Loved your video.
Do you sell liquid culture? And if so, what would it cost for the moral culture?
I am just getting into mycology, and the moral is my target.
I am also getting into different varieties of oyster, and medicinal as well.
The only thing i don't have is the flow fan and the moral culture, but i am working on that.
Thank you for your time and your video, very knowledgeable my friend!
check out our ETSY store FreshFungi! MUSHLOVE
Where did you buy the extractor? Mushlove
Lions Mane Equipment and Supply in Denver 👍
When will it be back in stock
Hi Gary. I didn't see a cap on that media luer lock connector and I did not see you spray it with alcohol before connecting your sterilized pipette. Any insight into this. Was something missed in editing? Thanks.
it was in the flow hood and this was before matt added caps to that contraption- there is a back flow valve so any fluid only went from the check valve forward. Not ideal but it worked
@@FreshfromtheFarmFungi thanks for the reply!
Back with another question for my Fungi Guru😊
Question number one when pouring agar into Petri dishes I'm assuming the steam on the lid is from my pouring it in too hot and I should let it cool more unfortunately I already started the process so I just turn the lids upside down for about 10 minutes and then flip them over. What negative effect does the moisture have inside the Petri dishes?
Second question and I'm sure you probably made a video of it already if so please just tell me the name and I'll go watch it LOL but the question was what is liquid culture made from and why can you store it so much longer than agar Petri dishes
Condensation isn’t a huge deal you can let them dry out in front of a hood - the LC is made from honey and water 👍 It’s a 3d matrix compared to a 2d plate so sustains longer growth
Thanks for all your videos. What do you think caused the contams for your Cordyceps LC? 90 minutes at 15 psi was too short? The slant culture? Honey? How do you keep from being paranoid and losing your mind trying to locate the source of the contamination? Would you have had more confidence if you hadn't been making a video. Are you confident the media extractor isn't picking up bacteria with that hanging port?
The contamination probably occurred from poor technique by me during inoculation of the culture into the LC (yeast or bacteria from my skin probably dropping into the vessel - just takes one viable cell to enter the bottle and multiply exponentially). The best way to find out where or when is to leave out blanks or let the LC rest at room temp for a few days before inoculating. It should remain clear if it’s sterile and will plate clean. The original culture was clean because the next round worked fine. There is always human error in mycology however small or rare, which requires a lot of patience and monitoring and trial and error 👍🏻 MUSHLOVE
@@FreshfromtheFarmFungi could be systemic??? Your technique looked good.
Is that a check valve on the tubing that is dangling off the cap? the second fitting from the end? is that how it stops the liquid from getting contamination? Been trying to hit them up to buy one but no response...
Yes it is a .22 micron filter - they have been working hard to get their store online with all the recent events and he is going to be making some sort of kit in the future - everything is in the "prototype" phase and we are open to taking suggestions! MUSHLOVE
@@FreshfromtheFarmFungi That's the air intake side right? I mean the side where you withdraw the liquid. That has a filter too? or how is it keeping air from backflowing inside?
@@shanesmith89 thats a one way check valve
Hello. Nice video. So I wonder if you keep the bottle cap tighly close, not allowing gas trasnfer during lc incubation?
I screw it tight and make sure there is headspace for fresh air then when mixing in the hood take the cap off to allow for air exchanges. They make these lids with a filter patch as well
@@FreshfromtheFarmFungi I see. so you mean enough headspace is the key correct? I would like to try growing a smaller culture in 50 mL tube as a seed culture of bigger bottle. I guess it can be done. Thanks
@@FreshfromtheFarmFungi if the tube screw tight for several days with a lot of head space, (80%), do you think it would be okay to leave it unopened for several days until the cultures fully grown?
how can i alcohol extract mycelium, lions mane?
What is the syringe you are using to draw out the liquid? What mls but also tip size (1/4?) What are the complete specs on the glass pipette???????
I'm not sure it came with the setup - Lion's mane equipment and supply store in Denver has the info on that
Hi Gary I am starting to use these media extractor bottle setups and have a question- at 19:00 in you take OFF the lid to re sterilize- why?? Isn't the point of these lids to have to NEVER take the lid off?
Are you concerned about the liquid in that extraction tube sitting there? I am wondering because when I PC my bottles with these lids on (and not screwed TIGHT just loosely on) that tube will fill up, and I am wondering if I should be concerned with that tube sitting full like that long term?
Im going to do a followup video on these after a few years use - I took the cap off to clean it thoroughly so I could run another batch through it - they get clogged with mycelium over time and it’s better to stay ahead and keep it clean before it gets plugged. Sitting full shouldn’t effect it as long as it was sterilized properly 👍
@@FreshfromtheFarmFungi right on man I look forward to seeing that, and thanks for the quick reply! cheers!
Love the importance of QC. What’s the rate of liquid cultures that have failed at the QC point?
It varies throughout the year because of environmental patterns but usually 3-10% is average for me
Is there a reason that you need to use these media bottles for LC instead of just using ball jars?
no but the openings are smaller, so less chance for contams
How come the grain jars you don’t have the self injecting port or a gas exchange filter disk?
the lids flipped like this and the threading is enough space for fresh air exchange
is a really good video ! Can ibuy them in Europa?
try reaching out to the store they may ship internationally
4-5 days of cooling to room temp?
Don't have to speak directly into mic, 20 something in, almost blew my speakers lol. Thanks so much for the help, though. Can't wait to get some started myself.
yes the audio on these older videos was bad Im going to put captions in this winter but the newer videos are better ha
I’ll modify an autoclavable handsoap bottle. Thanks. 😁
cool idea! innovation at its finest 🍄❤️
Fresh from the Farm Fungi LLC Ditto. 😁
Is the blood agar required or could you do the QC with potato flake agar?
potato is fine
Can you please specify the types of QC agar you are using their names I cannot transcribe and find of hardy s website.
th-cam.com/video/iHYBu3rXHes/w-d-xo.html
What time of year do you inoculate your yard?
sporadically throughout the whole year - at least once a week with some fresh mycelium or spore slurry
How Mach time on proses airator liquid culture
Hey that part of the granular look was new for me.
When I stir my liquid cultures made at home with a pressure cooker and a simple still air box they don't look cloudy but there are big chunks in it instead so finely distributed as it looked in your other jar.
Is that in general a sign of contamination or is that dependent on the type of mushroom?
Since I stir my cultures rarely might that also be a reason?
And for how long did you stir before it was that homogeneous?
May I buy MOREL liquid spawn? Can you send to THAILAND? If posible please give me the price+shipping and payment method such as Pay pal or Western union. Thanks
You meant grain spawn......also a Injektion port on the jars would be good
what is the green and red QC plate you got from diagnostics ?
sorry answering own question - please see this video for full description
th-cam.com/video/iHYBu3rXHes/w-d-xo.html
Yes - the green is HE and the red is SBA - both are commonly used screening QC plates
Where can I find transfer caps with filters like that!
Lions Mane Equipment and Supply has them 👍
So you don't need a gas exchange filter to colonize LC?
it helps but it’s not necessary
how do you know what temperture you are at with the pressure cooker,, says, 250F..
thermometer
If you were to grow out the contaminated mycelium would the mushrooms be poisonous??
My talk on contamination - treat mushrooms like meat and cook thoroughly m.th-cam.com/video/ssxdhydPYos/w-d-xo.html
Bees 🐝 and mushrooms 🍄 ❤️
I'm working on getting my family started on both. Can't wait to try using our own honey in our liquid cultures one day!