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Reviving H20 suspended mycelium


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#1 Needles

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Posted 04 December 2016 - 12:50 PM

There hasn't been much going on in this part of the site for awhile so I thought I would share a experiment with you.
I have been working with storing cultures in sterile water for about four years now. I will start with a monoculture on agar.image.jpeg
When the culture has grown out on the plate the mycelium is scraped off image.jpeg image.jpeg
Once harvested the mycelium is added to sterile water and blended image.jpeg after the mycelium has been shredded it is extracted with a sterile syringe then added to sterile bottles for storageimage.jpeg after the venders supply of bottles ran out I started looking at other options. I came across this septa bottle image.jpeg it's 40ml, a little larger than I like but seems to work ok.

My syringes are cooling in the PC now so later tonight I will inoculate two quart jars of grain for each species. image.jpeg image.jpeg most of these cultures are two years old but a couple are almost four now.
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#2 Microbe

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Posted 04 December 2016 - 06:46 PM

Needles, you and i are about to start hanging out, just an fyi!
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#3 Needles

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Posted 04 December 2016 - 07:16 PM

Needles, you and i are about to start hanging out, just an fyi!


I have learned so much from your agar work already. Looking forward to learning and sharing with the hybrids too.....
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#4 Needles

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Posted 05 December 2016 - 11:44 AM

It's on... Time will tell, it usually takes a week to start seeing mycelium recovery.image.jpeg
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#5 Needles

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Posted 15 December 2016 - 01:30 PM

i checked on the jars today and all but three are showing signs of recovery and growth image.jpeg
I mixed up a nutritional broth and added liquid innoculent for a fermentation tek. Hopefully it will give the older storage mycelium a jump start. image.jpeg the broth will be left on the magnetic stirrer for at least three days on a slow speed before a extraction and transfer will be made.
Today I am also experimenting with a reishi culture. I blended a fresh culture and injected three jars of rye with 5cc each of liquid innoculent. I also added 15cc to 150ml of nutritional broth. After three or four days I will inoculate three more jars of rye with broth and note any growth difference.
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#6 Tenderfoot

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Posted 15 December 2016 - 09:10 PM

Very cool lab set-up!!  Nice glass syringes and some choice strains...

 

This is old school!  Nice thread... I was always under the impression that there was to be no airspace in such jars.  Have you noticed any loss of vigor over time under such conditions in the past?  Strains can reportedly be preserved for years with this method.  Nice to see you revive it on the boards. 

 

Looking forward to this grow log unfold...


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#7 Needles

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Posted 16 December 2016 - 12:37 AM

Very cool lab set-up!!  Nice glass syringes and some choice strains...
 
This is old school!  Nice thread... I was always under the impression that there was to be no airspace in such jars.  Have you noticed any loss of vigor over time under such conditions in the past?  Strains can reportedly be preserved for years with this method.  Nice to see you revive it on the boards. 
 
Looking forward to this grow log unfold...

Thanks Tenderfoot, I'm still learning about this myself as time goes on. Not sure about air being a Issue but if there is extra nutrition like agar the mycelium tends to start growing on the surface of the water. I have also added a few other strains that were not in the original group of cultures. My plan is to get the jars colonized and then transferred to agar. Plus I want to try to get some of them to fruit....
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#8 Needles

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Posted 19 December 2016 - 01:03 PM

After five days my reishi jars are starting to look real goodimage.jpeg
I was also very surprised to see this growth image.jpeg that jar has less than 12 hour with 5cc of broth in it. Now the broth was made three days ago but the recovery on gain is amazing.
My turkey tail jars are totally recovered and most others are catching up. I will try to post a lineup photo of this project soon....
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#9 Needles

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Posted 25 December 2016 - 02:41 PM

A few of the jars are ready to be transferred back to agar plates today image.jpeg there are still two species that have not shown any growth good or bad so they will go on the shelf for a few more weeks. Today was the first day I saw growth in the King oyster jars, that was exciting as I didn't want to loose that one. There will be five more new species to start tomorrow and a few of these to go into sawdust bags.
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#10 Microbe

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Posted 26 December 2016 - 05:57 PM

What is causing some of those jars to show green on top of the grain? Im assuming it is something behind the jars.
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#11 Needles

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Posted 27 December 2016 - 12:42 AM

What is causing some of those jars to show green on top of the grain? Im assuming it is something behind the jars.


It's the lid on the totes. Fell across a bunch of sleeves of petri plates... I keep them in totes and the lid is green.

Everything is looking good, should have fresh mycelium from all those jars real soon..........
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#12 Sidestreet

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Posted 27 December 2016 - 05:56 AM

Are you going to fruit all of those, too?  :smile:

 

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Edited by Sidestreet, 27 December 2016 - 05:57 AM.

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#13 Needles

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Posted 27 December 2016 - 12:17 PM

Are you going to fruit all of those, too?  :smile:
 
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I would like to at least try to grow all these out. Some of these jars will be grain masters and others will go on agar to be spawned later this summer for fall fruiting. They are all wood lovers, I am waiting for a delivery of wheat bran to make up sawdust bags. Should have bags ready of the spring fruiting strains in a few weeks.......
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#14 back2dirt

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Posted 30 December 2016 - 01:27 AM

nice work


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#15 GLP

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Posted 30 December 2016 - 08:12 AM

Nice work needles, I especially like your little blender. I should have asked for that at Christmas, now I will have to wait for my birthday to ask the Mrs. for such a gift. Keep us informed on your progress.


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#16 Needles

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Posted 30 December 2016 - 09:25 AM

Nice work needles, I especially like your little blender. I should have asked for that at Christmas, now I will have to wait for my birthday to ask the Mrs. for such a gift. Keep us informed on your progress.

Check your local thrift stores for vintage Waring blenders. Their blades can easily be disassembled and made to fit a mason jar lid.

As far as progress, I only have a few more jars to transfer to agar. I think it's getting a bit out of control lol....image.jpeg
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#17 back2dirt

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Posted 30 December 2016 - 07:00 PM

post-136520-0-47798600-1480873087.jpeg

 

what's the round black piece on lid? or is that the other side of the blade?


Edited by back2dirt, 30 December 2016 - 07:02 PM.


#18 Needles

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Posted 30 December 2016 - 09:01 PM

what's the round black piece on lid? or is that the other side of the blade?


The black piece is what spins the blade, it is called a pinwheel style blender. It's threaded all the way through the lid. I used jet engine gasket material with stainless steel washers to make the seal. It can take the high temperatures of an autoclave with no leaks. There is another style of blender that is a post. It is a four sided motor that has a six sided base for the blade. They both work well, I just keep one style (pinwheel) so I don't need two different motors.......
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#19 GLP

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Posted 01 January 2017 - 01:09 PM

Can you explain the workflow in a little more detail please, I am assuming you have not PC'd your blender, so are you working with a flowhood and just wiping the blender down? Questions in my head also pop up with the transfer of lids from the blender lid to a PC'd lid with syringe port. I have search on blender slurry but still have a few holes in the details for my engineers brain to follow. :tinfoil:


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#20 Needles

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Posted 01 January 2017 - 04:44 PM

Can you explain the workflow in a little more detail please, I am assuming you have not PC'd your blender, so are you working with a flowhood and just wiping the blender down? Questions in my head also pop up with the transfer of lids from the blender lid to a PC'd lid with syringe port. I have search on blender slurry but still have a few holes in the details for my engineers brain to follow. :tinfoil:

A while back I posted this thread https://mycotopia.ne...th-of-it/page-4 it will show you step by step on the blender lid build.

Using a half pint large mouth jar seems to work the best. About half the jar is filled with water then with the blender lid screwed on it is PC sterilized for a hour. You can't rush the cooling down process or you can risk breaking the jar. Transferring mycelium to the jar is done in a flowhood. I keep the blender outside of the hood but I do sanitize it before hand. I run the blender on and off about five times, that will let the mycelium settle before blending again. The jar is then wiped down again with isopropyl alcohol befor it is brought back into the flowhood for extraction with a sterile syringe.
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