88 replies to this topic

[Needles]

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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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.

[raymycoto]

 

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]

 
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.

[av8or]

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......

[Microbe]

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.jpegimage.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.jpegimage.jpeg most of these cultures are two years old but a couple are almost four now.

Needles, i think you should update this thread, a lot of info and results to be shared since you posted this. Do it man.....do it.....do it.....do it!

[Needles]

Yes Microbe your right... my research in this field continues with a vengeance. It’s been eight years ago when I bottled my first culture. I placed a bottle in cold storage and one in the dark at room temperature. The bottle in cold storage was warmed for three days and 5ml of liquid inoculation was administered to a quart of sterile rye grain. After a week of no obvious recovery I had added five more milliliters to a nutritious broth of light malt mixed 40 g to 400 ml of distilled water and placed on a magnetic stir plate. Then set at a low speed and left for another week then once again to a quart of grain. After three days on grain I noticed a visible colony recovering. That was very exciting I checked the other jar and it had signs of recovery too. I’ve also placed a drop on a slide and inspected under oil immersion microscope. There was a considerable amount of bacteria living alongside the blended mycelium. The jars are still in the middle of colonization so I’m not sure about their health. 
As time permits I will update this thread and my eight year old mycelium recovery and other projects with visual photos of equipment that I’ve upgraded to....

Edited by Needles, 10 February 2021 - 08:01 PM.

[Microbe]

Yes Microbe your right... my research in this field continues with a vengeance. It’s been eight years ago when I bottled my first culture. I placed a bottle in cold storage and one in the dark at room temperature. The bottle in cold storage was warmed for three days and 5ml of liquid inoculation was administered to a quart of sterile rye grain. After a week of no obvious recovery I had added five more milliliters to a nutritious broth of light malt mixed 40 g to 400 ml of distilled water and placed on a magnetic stir plate. Then set at a low speed and left for another week then once again to a quart of grain. After three days on grain I noticed a visible colony recovering. That was very exciting I checked the other jar and it had signs of recovery too. I’ve also placed a drop on a slide and inspected under oil immersion microscope. There was a considerable amount of bacteria living alongside the blended mycelium. The jars are still in the middle of colonization so I’m not sure about their health.
As time permits I will update this thread and my eight year old mycelium recovery and other projects with visual photos of equipment that I’ve upgraded to....

Im pretty confident that bacteria does not pose any risk to the mycelium and might very well be part of the environment within and surrounding fungal hyphae, sometimes known as the mycosphere. It is a symbiotic relationship and the bacteria resides inside the hyphae and are released when the mycelium is blended as the hyphae tips are removed, which is why blended mycelium may take 2 weeks or more to recover and begin to grow. I did a gram stain a few years ago and i forget what the results were, but the culture performed as normal or up to my expectations. I will bet you 1 flow hood, 2 billy goats, and one bushel of Egyptian Cotton Pine leaves, that your jars are going to be fine. Keep us updated and post pics brother.

Edit: i understand that a gram stain alone is not sufficient in helping to identify the bacterium or bacteria present. Can you use your electron magnetic scope to get a exact id on what is present?

Edited by Microbe, 10 February 2021 - 08:35 PM.

[Arathu]

 

Yes Microbe your right... my research in this field continues with a vengeance. It’s been eight years ago when I bottled my first culture. I placed a bottle in cold storage and one in the dark at room temperature. The bottle in cold storage was warmed for three days and 5ml of liquid inoculation was administered to a quart of sterile rye grain. After a week of no obvious recovery I had added five more milliliters to a nutritious broth of light malt mixed 40 g to 400 ml of distilled water and placed on a magnetic stir plate. Then set at a low speed and left for another week then once again to a quart of grain. After three days on grain I noticed a visible colony recovering. That was very exciting I checked the other jar and it had signs of recovery too. I’ve also placed a drop on a slide and inspected under oil immersion microscope. There was a considerable amount of bacteria living alongside the blended mycelium. The jars are still in the middle of colonization so I’m not sure about their health.
As time permits I will update this thread and my eight year old mycelium recovery and other projects with visual photos of equipment that I’ve upgraded to....

Im pretty confident that bacteria does not pose any risk to the mycelium and might very well be part of the environment within and surrounding fungal hyphae, sometimes known as the mycosphere. It is a symbiotic relationship and the bacteria resides inside the hyphae and are released when the mycelium is blended as the hyphae tips are removed, which is why blended mycelium may take 2 weeks or more to recover and begin to grow. I did a gram stain a few years ago and i forget what the results were, but the culture performed as normal or up to my expectations. I will bet you 1 flow hood, 2 billy goats, and one bushel of Egyptian Cotton Pine leaves, that your jars are going to be fine. Keep us updated and post pics brother.

Edit: i understand that a gram stain alone is not sufficient in helping to identify the bacterium or bacteria present. Can you use your electron magnetic scope to get a exact id on what is present?

 

I definitely agree ^^^^^

 

A

[Needles]

Even our favorite fungus has a normal flora of bacteria. It may not show up on a transfer to agar but still there. @ Arathu we will have to bring the yellow hen into this thread and show how bacteria can have a symbiotic relationship with fungus. I’ll dig up some pictures.....

[Arathu]

Hopefully......We will get it to fruit......you've done all the work so far....

 

I have oak soaking in a nutrient bath right now actually......

 

A

[Microbe]

I can't edit my post anymore....i was just reading it........but i meant that 'specific' bacteria under the scope. Bacteria in general is very bad for what we do. Shit i wish i can edit that just in case someone reads it 5 years from now

[Microbe]

Even our favorite fungus has a normal flora of bacteria. It may not show up on a transfer to agar but still there. @ Arathu we will have to bring the yellow hen into this thread and show how bacteria can have a symbiotic relationship with fungus. I’ll dig up some pictures.....

Exactly. I remember when you were getting pissed off about looking at your blended cultures and seeing bacteria.....you couldn't figure it out. I told you its normal, your not doing anything wrong as you have the cleanest freaking home mycolab on the planet including gadgets with that PSU hook up. All fungi require the most abundant organism on the planet to 'make it' to the end.

Imma going to make a post here soon about bacteria and mushrooms......you know my randoms thread talks about it a lot. This will blow your mind though brother

[rockyfungus]

Assuming fungi are similar to ourselves, or any organism for that matter.

Most of us are more bacteria/virus then human anyways ;)

 

"In any human body there are around 30 trillion human cells, but our microbiome is an estimated 39 trillion microbial cells including bacteria, viruses and fungi that live on and in us."

From a random website, don't wanna dig up a real scholarly article.

[Needles]

Assuming fungi are similar to ourselves, or any organism for that matter.

Most of us are more bacteria/virus then human anyways ;)

 

"In any human body there are around 30 trillion human cells, but our microbiome is an estimated 39 trillion microbial cells including bacteria, viruses and fungi that live on and in us."

From a random website, don't wanna dig up a real scholarly article.

we all need that symbiotic relationship. I have read where the average person has five pounds of bacteria living on and in them. Also by inspecting tiny insects under the scope and cameras equipped with 10x objectives and have found living Bactria in their flora. I believe that bacteria could easily take over the organism but if it did it would also parish.

[Needles]

I have a few pix from the last recovery attempt, it is a MS culture of Azur from 2013 plus a bottle of Hypsizigus ulmarius from this year plus lions mane from today.

 

This is a photo of the Azur culture on grain. After using a sharpie to label on painters tape I found that isopropyl will wash the labeling away. Using a grease pencil or china marker works great and will not wash off. I have recently switched to Avery clear laser jet labels and have good luck with them.

 

 

A bottle from 2015 using a sharpie  and a recent bottle using Avery labels 

 

This photo is of a lions mane culture that was grown out on a 150mm Pyrex plate. Mycelium was harvested right under the colony a small amount of nutritious agar was taken with it and blended. I feel that including a small amount of agar is beneficial in the recover process. Like giving it a bit of a foundation to hold on to.

 

[sandman]

Needles, your pics make me feel funny in my underpants area

 

You got all the neato wizbangs

 

Lots of great info on this thread

Edited by sandman, 13 February 2021 - 08:56 PM.

[Needles]

A photo of the storage bottles with Avery labels applied. So far the best method of identification to keep track of strain and year... 

 

 

[sandman]

stop, stop...I can only get so hard

[Arathu]

Beautiful stuff brother.........beautiful....

 

I think I'm going to get a load of logs this year.......

 

And make some plugs......

 

A

[Needles]

Plugs? Did someone mention dowels.... Pleurotus Salmoneostramineus, Pholiota Adiposa and Pleurotus Cystidiosus coming up...