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Reviving H20 suspended mycelium


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#41 Heirloom

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Posted 13 May 2017 - 09:45 PM

Have you ever used activated carbon to revive mycelium. The reason I ask is I have heard it from a knowledgeable cultivator to germinate " hard to germinate spores"?

Looking for a blender so I can fill these sterile 10ml serum vials, I was afraid the caps on amber dram bottles might melt in a pc. Will use syringes if needed for storage but vials are much easier to store due to size.

this thread is awesome makes the journey fun , got lots of fun to do with spores and cultures and acquiring lab equipment.

peace man

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Edited by Heirloom , 13 May 2017 - 09:48 PM.

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#42 Heirloom

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Posted 17 May 2017 - 12:29 PM

Thanks needles , now I can justify buying some flasks, beakers and a good magnetic stirrer for a hobby lab.
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#43 Needles

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Posted 20 May 2017 - 07:46 PM

Thanks needles , now I can justify buying some flasks, beakers and a good magnetic stirrer for a hobby lab.


Awesome.... when you get your flasks, order 500ml Pyrex (I like the ones from Germany) with threads and caps. You'll love them for making agar. I drilled 1/4" holes in the caps and added a synthetic filter. They work great....
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#44 Arathu

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Posted 23 May 2017 - 06:53 PM

I have to get some of these too and a mag stirrer ........and shelving.....and more sterilization capacity .........so says the dungeon master.............hahahahaha 

 

The list grows............

 

A


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#45 Kmmfarm

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Posted 04 June 2017 - 12:06 AM

Awesome show, I will definitely have to try some of your experiments for my self, will be watching keep up the good work.


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#46 Cigarsam

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Posted 04 June 2017 - 03:42 AM

Needles, excellent thread.
Thank you for sharing your knowledge.
Archive ^^...
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#47 sandman

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Posted 04 September 2017 - 11:41 PM

I have heard of using mineral oil or something similar in these little water jars for ultra long term storage. I guess it lays on top of the water and keeps the air off it. 

 

edit: maybe it was just plain mineral oil and no water I can't recall. 


Edited by sandman, 05 September 2017 - 12:01 AM.


#48 raymycoto

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Posted 24 January 2018 - 11:38 AM

I have a few questions and I'm interested in liquid culturing - I’m new and don’t want to clutter your thread, but you have great experience and I’m wondering about some things brought up on this thread:
 
Any thoughts on sterile water vs nutrient solution for storage? An analogy might be use use of refrigerated agar slants for long term storage.
 
The scientific article cited referred to a ‘cork punch’ used to extract mycelium from agar. I have had good luck with one of these sets - cheap, autoclavable (or I just flame and dip in sterile water to cool it in between samples). See attached image - can't get it to load inline unless it's a URL.
 
Blender question or warning - Blenders are indeed used to disrupt cell membranes to emulsify cells for analysis. Be careful, I guess about hitting the myc too hard with the blender. I thought about using a blender but then decided not to, but then I do think it’s a good idea to break up the myc to at least some extent for a liquid culture. What are your thoughts on this? Run blender briefly? Low speed? Modified blades?
 
I noted your malt conc was 10 gm per dl. I have read on this forum that perhaps 5 gm per dl was a “max” although I guess we are talking about grams % and not molarity or osmolarity. Malt, I guess, is a mostly a starch, which would have a large molecular weight and thus less osmolarity than, for ex., dextrose. The hazard with high conc might be osmotic effect on the cells (?)
 

 

Can you give a reference to the flasks with the screw tops? Have not heard of that. Erlenmeyer, I presume? 
 
Thanks for all your work.

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Edited by raymycoto, 24 January 2018 - 11:40 AM.


#49 Microbe

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Posted 24 January 2018 - 04:46 PM




I have a few questions and I'm interested in liquid culturing - I’m new and don’t want to clutter your thread, but you have great experience and I’m wondering about some things brought up on this thread:


Any thoughts on sterile water vs nutrient solution for storage? An analogy might be use use of refrigerated agar slants for long term storage.


The scientific article cited referred to a ‘cork punch’ used to extract mycelium from agar. I have had good luck with one of these sets - cheap, autoclavable (or I just flame and dip in sterile water to cool it in between samples). See attached image - can't get it to load inline unless it's a URL.


Blender question or warning - Blenders are indeed used to disrupt cell membranes to emulsify cells for analysis. Be careful, I guess about hitting the myc too hard with the blender. I thought about using a blender but then decided not to, but then I do think it’s a good idea to break up the myc to at least some extent for a liquid culture. What are your thoughts on this? Run blender briefly? Low speed? Modified blades?


I noted your malt conc was 10 gm per dl. I have read on this forum that perhaps 5 gm per dl was a “max” although I guess we are talking about grams % and not molarity or osmolarity. Malt, I guess, is a mostly a starch, which would have a large molecular weight and thus less osmolarity than, for ex., dextrose. The hazard with high conc might be osmotic effect on the cells (?)



Can you give a reference to the flasks with the screw tops? Have not heard of that. Erlenmeyer, I presume?


Thanks for all your work.


For long term storage in liquid media it cant have any O2 or Nutrients. If it has O2 then it will want to conduct cellular respiration which requires energy and thus will eventually starve to death. If there is nutrients then again it will want to conduct cellular respiration which requires O2, thus it will suffocate.

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.

The only O2 in the water will be disolved and minimal. Note the O2 molecule making up the H2O is not a available source of O2 for the mycelium just the free O2 molecules.

Simply stated the culture seases all metabolic functions which puts it a state that goes beyond hibernation and more like a antimated state of suspension.

Spores can be stored the same way. I posted a link somewhere around this site where a experiment was done with a dozen cultures of many species and over 90% were recorded at the 4 year mark when the experiment was concluded. Could they go longer? Absolutely. Some species may not keep as long or not at all but im certain Cubes will keep a very long time, probably a decade.

My buddy said Peter McCoy talks about it in his Rodical Mycology Book which i have yet to read after he gifted me a copy a few years ago(I have ADHD and cant focus very long) but this lead me to do some research and it is limited but there are some good studies out there if you dig deep enough.

I believe this can replace master slants because they require maintenance and although its slow, there is still metabolic activity thus the culture is aging and needs Fresh air exchange periodically.

Finally and not to get to far off topic, you can also use cryo- protectants such as a food grade propylene glycol. I dont have the chart handy but it doesn't take much to lower the freezing point of liquid to below freezing. I want to say 3% gets you down somewhere around 27° i think. Even though PG has antifungal properties, at that concentration it is not toxic and can actually be a food source. H8gher concentrations beyond 5% probably needs to have the culture rigorously exposed to it slowly upping the concentration but would be worth it if you can get the culture into sub zero temps without harming it.

The only down side to this and this speculation, im not sure this training will be passed on to subsequent generations. The PG being recognized as a food source probably would but that is irrelevant as the goal is to put it in a state where all metabolic activity halts so no eating allowed. The other and is fact, the culture needs to slowly and i mean very slowly brought back up to room temp. You can't just toss it in a incubator.

This IMO would be superior to all other long term storage methods and can easily be done at home with a deep freezer. I started a trial but abandoned it for some reason, maybe my LC contaminated i don't remember but it has not been forgotten.

Hyphae are single cell wall thick including the septa that bonds the colony or all the individual hyphae, it doesnt take much to destroy that infrastructure and happens frequently in many processes. The colony will repair itself and grow back together or begin to form individual colonies once put back into a nutrient media.

On the contrary, my favorite thing becauae I see both sides of most things, i can certainly see those individual hyphae that get obliterated not making it through the long term storage but keep in mind were not creating inoculate and you will see large strains of mycelium after the blending process. For simple conversation, even if only a few strains of mycelium survive, its successful because the goal is to bring in back to its normal metabolic functions then can be expanded back to create another healthy colony with X amount of mycelia mass.



@needles one of those BD 50' s , the flange at the top broke durung shipping. Thats the one I'm giving you when come through thanks for letting me throw a little info out there on the subject.....

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#50 Needles

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Posted 24 January 2018 - 09:42 PM

Good information brother Microbe. I'm thinking about the no O2 as my next step forward into long term, five to ten years of culture storage. Up until now it has been five years of studying this method. Definitely any extra nutrition like to much harvested agar can cause unwanted growth in the storage vessel. Last week I took a four year old MS culture that had been stored at room temperature and started the revival of suspended mycelium. This culture was in a 30 ml bottle with a portion of O2 from not filling 100% of the bottle. Using slow agitation for three days, the culture was kept in a nutritional broth fortified with a very small amount of gypsum and nutritional yeast. This liquid fermentation tech has proved to be incredibly viable with an unbelievable rate of growth.

My concern with using this storage technique has been with bacteria. I am still working on some further studies of this but with twelve cultures tested all twelve were positive for Cocci bacteria. I believe that the mushroom cultures I have been working with have a "natural flora" of living bacteria. Of the twelve, not one has been infected with a Bacillus, Duplo, Staff or Streptococcus. Even after inoculations of numerous master grain jars, none show any indications of bacterial infection but I know its there.

@Microbe, your welcome for the labware and I'm looking forward to you sharing that cracked 50 with me....
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#51 Needles

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Posted 24 January 2018 - 09:58 PM

I have a few questions and I'm interested in liquid culturing - I’m new and don’t want to clutter your thread, but you have great experience and I’m wondering about some things brought up on this thread:
 
Any thoughts on sterile water vs nutrient solution for storage? An analogy might be use use of refrigerated agar slants for long term storage.
 
The scientific article cited referred to a ‘cork punch’ used to extract mycelium from agar. I have had good luck with one of these sets - cheap, autoclavable (or I just flame and dip in sterile water to cool it in between samples). See attached image - can't get it to load inline unless it's a URL.
 
Blender question or warning - Blenders are indeed used to disrupt cell membranes to emulsify cells for analysis. Be careful, I guess about hitting the myc too hard with the blender. I thought about using a blender but then decided not to, but then I do think it’s a good idea to break up the myc to at least some extent for a liquid culture. What are your thoughts on this? Run blender briefly? Low speed? Modified blades?
 
I noted your malt conc was 10 gm per dl. I have read on this forum that perhaps 5 gm per dl was a “max” although I guess we are talking about grams % and not molarity or osmolarity. Malt, I guess, is a mostly a starch, which would have a large molecular weight and thus less osmolarity than, for ex., dextrose. The hazard with high conc might be osmotic effect on the cells (?)
 
 
Can you give a reference to the flasks with the screw tops? Have not heard of that. Erlenmeyer, I presume? 
 
Thanks for all your work.


Microbe seemed to answer a lot of the questions you were asking. You were asking about flasks and lab glass. I'm a Pyrex freak but I'm sure most flasks will work. I have found that some of the less expensive glass will not be as good as USA or Germane Pyrex.
I use a online auction site for almost everything I purchase. Try key words screw top erlenmeyer flask pyrex

I hope to be able to post more on this subject and share my method of mycelium storage. So I hope to see you around, welcome to Topia.......
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#52 sandman

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Posted 25 January 2018 - 11:29 AM

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.



#53 Needles

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Posted 25 January 2018 - 11:57 AM

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.


I'm feeling your pain for loosing cultures. I think most of us have been there too. After working so hard to germinate, isolate, grow and clone it's hard to loose a culture like that.
Were you trying to revive to agar? Do you still have your slants? If you have a magnet stirrer, try a malt broth for a few days on slow speed. I found amazing results from using a fermentation tech like that. Hopefully you can get the same results......
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#54 sandman

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Posted 25 January 2018 - 12:34 PM

I did put it into malt LC and also on agar. 0 recovery, pretty strange. 


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#55 Microbe

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Posted 28 January 2018 - 09:14 AM

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?

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#56 sandman

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Posted 28 January 2018 - 10:26 AM

No I was trying to figure it out logistically. It's actually 2 thin sticks of popsickle stick material but they are more like 4 tooth picks in size. It would be easy enough to clip it with some scissors or clippers but I'm worried the clipping would fly away. Maybe i could just shave off a part with my scalpel?



#57 sandman

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Posted 28 January 2018 - 10:27 AM

I guess I could just lay the whole stick in there ehh, duh



#58 Microbe

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Posted 28 January 2018 - 10:41 AM

No I was trying to figure it out logistically. It's actually 2 thin sticks of popsickle stick material but they are more like 4 tooth picks in size. It would be easy enough to clip it with some scissors or clippers but I'm worried the clipping would fly away. Maybe i could just shave off a part with my scalpel?

You do want to try to expose the inner part of the stick to the agar surface. I'd you soaked the sticks in dextrose solution, there is a excellent chance that there is some viable hyphae in the center. I always soak mine for that reason. Simply pouring nutrient agar in a slant wont allow for the nutrients to penetrate the stick because the agar solidifies so quickly. I would try to cut in 3rd or more if possible then lay them down on agar plates prepared a little wetter then normal. Hopefully you will get some myc jump off one of the ends. The more cuts you can make the better. I would fit as many in 1 plate as you can, no need for multiple plates and as soon as you get some colonizing myc then put it on a regular hydrated agar with a little nutrient spike around 5%. Good luck.

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#59 sandman

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Posted 28 January 2018 - 12:16 PM

The sticks and slants were prepared "no pour" style and were PC'd with the agar and sticks in the tubes so they probably got a good penetration. I will try to pull a stick out and split it in 3 strips like a banana sorta and lay those 3 strips in a dish and see how that goes.


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#60 Microbe

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Posted 28 January 2018 - 12:23 PM

The sticks and slants were prepared "no pour" style and were PC'd with the agar and sticks in the tubes so they probably got a good penetration. I will try to pull a stick out and split it in 3 strips like a banana sorta and lay those 3 strips in a dish and see how that goes.

Cool man. I typically do i no pour also but i still soak the sticks out of habit. Never thought about the pressure pushing the nutrient agar into the stick. My sticks are so water logged the agar wont penetrate much. Something to look into for sure, mycelium probably has a better chance of surviving if the agar penetrates the wood. When you make your slants what nutrient % do you run?

Something i have been thinking a out also is you mentioned the mycelium looks healthy it just won't colonize is that correct?

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