Paradox
©
Fisana

Jump to content


Photo

Reviving H20 suspended mycelium


  • Please log in to reply
63 replies to this topic

#61 Needles

Needles

    Locked and Loaded

  • Black VIP
  • 1,240 posts

Donator


Awards Bar:

Posted 28 January 2018 - 01:59 PM

I need to do something along these lines, just lost a few slants of nice old cultures only 8-12 months old. They are just dead. They look good but the mycelium will not recover when I transfer a piece out. They were 25mm tubes with a piece of Popsicle stick and MEA.

Have you tried putting the stick on agar?
Sent from my LGLS992 using Tapatalk

Good idea to try reviving those cultures.
Give them time too. I have had cultures take over three weeks to start to recover on agar. I thought they were dead, just put the plates on a shelf and one day they were growing again.
  • Arathu and Microbe like this

#62 raymycoto

raymycoto

    Mycotopiate

  • Gold VIP
  • 273 posts

Awards Bar:

Posted 28 January 2018 - 05:27 PM

 

To get around both of these you sterilize a jar, tube , etc of distilled water with no GE hole and this will create a vaccum. Then you harvest the mycelium from a agar plate leaving behind the agar and blend it thank you needles for my blender lids and motor, then you draw into the syringe and when you inject the container with the sterile distilled water under vacuum, it pulls in the mycelium water, which also needs to be distilled as i forgot to mention, the contents will be pulled in by the vacuum.

Excellent thread, lovin' it.

 

How can you assure there are no nutrients available to the myc? There may be some agar present from the initial harvest. Perhaps wait some time (a few days, week?) with the suspension in the sterile water prior to refrigeration -> let the myc eat up any remaining nutrients. But . . . if done in a vacuum, then there is no O2 for burning the nutrients. Maybe nutrient and no O2 is bad (wondering if myc has any anaerobic metabolic options like with animals.

 

Or do we just assume that we did not pick up much agar with harvesting of the myc?

 

I have an idea and wonder what you think about it. I have a professional commercial vacuum chamber sealer that can seal liquids. I gets to a really high level of vacuum, like < 1 mm Hg, then seals under vacuum and better that what you will get with a steam seal in a jar.  It seals with heat in tough plastic bags.

 

I know what you are thinking. It's not anything like a cheap vacuum sealer. This thing weighs 80# and is around $600 but, btw, is an awesome kitchen and lab tool you never knew you needed. Unlike an external vac sealer, it can do liquids.  Anyway, getting off topic but this is a good intro to the machine.

 

The sealer bags are autoclave tolerant. I imagine you could 'clave some bags or just do a chemical sterilization, then blend and seal some myc and refrigerate. I'll take some pics and maybe do a demo for kicks.  The myc would be at atmospheric pressure in a flexible bag but sans any gas in the bag or the liquid for that matter. The vacuum chamber extracts gas from the liquid as well to some extent (exp with the longer vac cycles available). And the bags are not gas permeable.

 

Can you give any references etc for preservation of myc under nutrient free medium and vacuum. Would be a cool but really lengthy (!) experiment to do - compare long term storage techniques and conditions. Could do 4 groups - Vacuum or not combined with nutrient or not.

 

Thanks again.


Edited by raymycoto, 28 January 2018 - 05:29 PM.

  • Needles likes this

#63 Needles

Needles

    Locked and Loaded

  • Black VIP
  • 1,240 posts

Donator


Awards Bar:

Posted 28 January 2018 - 06:22 PM

 
To get around both of these you sterilize a jar, tube , etc of distilled water with no GE hole and this will create a vaccum. Then you harvest the mycelium from a agar plate leaving behind the agar and blend it thank you needles for my blender lids and motor, then you draw into the syringe and when you inject the container with the sterile distilled water under vacuum, it pulls in the mycelium water, which also needs to be distilled as i forgot to mention, the contents will be pulled in by the vacuum.

Excellent thread, lovin' it.
 
How can you assure there are no nutrients available to the myc? There may be some agar present from the initial harvest. Perhaps wait some time (a few days, week?) with the suspension in the sterile water prior to refrigeration -> let the myc eat up any remaining nutrients. But . . . if done in a vacuum, then there is no O2 for burning the nutrients. Maybe nutrient and no O2 is bad (wondering if myc has any anaerobic metabolic options like with animals.
 
Or do we just assume that we did not pick up much agar with harvesting of the myc?
 
I have an idea and wonder what you think about it. I have a professional commercial vacuum chamber sealer that can seal liquids. I gets to a really high level of vacuum, like < 1 mm Hg, then seals under vacuum and better that what you will get with a steam seal in a jar.  It seals with heat in tough plastic bags.
 
I know what you are thinking. It's not anything like a cheap vacuum sealer. This thing weighs 80# and is around $600 but, btw, is an awesome kitchen and lab tool you never knew you needed. Unlike an external vac sealer, it can do liquids.  Anyway, getting off topic but this is a good intro to the machine.
 
The sealer bags are autoclave tolerant. I imagine you could 'clave some bags or just do a chemical sterilization, then blend and seal some myc and refrigerate. I'll take some pics and maybe do a demo for kicks.  The myc would be at atmospheric pressure in a flexible bag but sans any gas in the bag or the liquid for that matter. The vacuum chamber extracts gas from the liquid as well to some extent (exp with the longer vac cycles available). And the bags are not gas permeable.
 
Can you give any references etc for preservation of myc under nutrient free medium and vacuum. Would be a cool but really lengthy (!) experiment to do - compare long term storage techniques and conditions. Could do 4 groups - Vacuum or not combined with nutrient or not.
 
Thanks again.

First off I gotta say I'm sold on the vacuum sealer. A little pricey but could pay for itself with the bags alone. I came across boxes of glass vacutainer tubes, no caps but I have found expired ones online. I could see that being the answer to vacuum prepping for storage bottles.
I have cultures living in water now for five years. I'm sure that there was extra agar that went in plus I always just filled bottles about 2/3 to 3/4 full and no vacuum. The only trouble I had was when trying to store LC or nutritional broth with mycelium. Cultures would grow in the bottle.
If you don't have blender bags, it was posted here about just using a syringe to extract from a petri plate. I would use a blunt tip needle for that. Thanks again for posting the link to sealer, btw welcome to Topia.
  • Salem likes this

#64 av8or

av8or

    Bump Master

  • VIP
  • 77 posts

Awards Bar:

Posted 08 July 2018 - 08:37 PM

What a great read. Every time I think I found the best thread out there, another one comes up and kicks me in the but. Thank you all for your years of dedication to the art and freely passing it on to others to pass on, Is this a great site or what......






Like Mycotopia? Become a member today!