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trying to rescue an infected culture.


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#1 Needles

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Posted 14 March 2016 - 09:28 PM

Going through my shelf of cultures I came across this guy. image.jpeg
It was total neglect on my part and ended up with a pool of condensation and some kind of black mold. Rather than to open this around my work station I decided to mix up a batch of agar and add H202 and make transfers in the kitchen. I have been working with this stuff for a while and have read mixed reviews. It's not 100% when it comes to no contamination but I have had more good than bad luck using it. I can't say for sure if this will work or if the culture is even alive or not but its worth a try. buy studying others reports and formulas I have come up with a recipe of my own that seems to work well. The key is to use materials that are not H202 degradable. That is if your substance foams when it comes in contact with hydrogen peroxide it is H202 degradable and should not be used. No need for a large batch as I like to use 60x15mm Petri dishes for this type of experiment. These amounts are for 250mm of agar...

5 grams agar agar
.5 gram nutritional yeast
1 gram flour (I like to use milo)
.5 gram powdered wood pellets
.5 gram hard pet food ground
1 gram malt sugar
.5 gram dextrose

Mix well and sterilize at 15 psi for thirty minutes. Cool just before anger starts to coagulate mix in 1.5 ml of hydrogen peroxide image.jpeg
Using a regular kitchen cleaner wipe down your counter surface. I did wear gloves and a mask but only because I was close to the plate and didn't want to chance inhaling a large amount of spores. Other than that everything was done out in open air. image.jpeg Even the plates were left open for a while to cool.image.jpeg
Now just to wait and see what happens. This works great for cloning too. It just takes the mycelium a while to take off and can give the plate a chance to contaminate....
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#2 wharfrat

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Posted 14 March 2016 - 10:08 PM

excellent recipe and write up brother needles. I have used this recipe courtesy of needles and i can verify it's effectiveness, at least for what i was working with, a dirty foraged clone.


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#3 Needles

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Posted 18 March 2016 - 01:02 AM

This is a very cool way to incubate a few plates, the Ranco keeps the temp within a few degrees with minimal amount of energy. Lid down and this extra heat helps to keep the plates from forming to much condensation. Whenever cloning or making isolations it's best to have as little moister around your sample as possible.image.jpeg image.jpeg
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#4 Needles

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Posted 19 March 2016 - 11:33 AM

This window shade worked out even better than I was hoping for. It's heavy duty bubble wrap design and the way it folds makes it a perfect fit for a six inch heating pad making it a nice little portable incubater.....

#5 Needles

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Posted 19 March 2016 - 11:54 AM

I lost a couple plates to the mean green and that was growing right on the isolation. This was the best looking plate that grew, so now it's time to grab that leading edge and expand to normally sterilized agar. back to 100mm plates for the next step plus a few extra days to see how it grows.
image.jpeg image.jpeg
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#6 Needles

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Posted 29 March 2016 - 07:45 PM

I took a few more photos to share. First one is a shot of a culture that is still on H202 agar. Check out around the ten o'clock position, there is a very small colony of that black mold. It didn't grow on the agar but on the mycelium.image.jpeg the next shot is another micrograph of a transferred piece of mycelium onto MYA that was died with black food coloring. image.jpeg the next one is a isolate to MYA with blue food coloring. image.jpeg this strain has been isolated to PDA and should be ready to go to grain sometime next week......
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#7 truMushrooms

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Posted 29 March 2016 - 10:23 PM

Looking good Needles, nice write up too

I've never tried using the antibiotic agar, sounds like it could come in handy sometimes 

What culture is the one you've been working on? Looks like oyster myc 


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#8 Needles

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Posted 30 March 2016 - 12:26 AM

Looking good Needles, nice write up too
I've never tried using the antibiotic agar, sounds like it could come in handy sometimes 
What culture is the one you've been working on? Looks like oyster myc

Thanks for checking it out, this stuff works great for cloning too. The culture is P. weilli it's a wood lover that's why I added the crushed wood pellets. Hope you get a chance to give it a try and you get good results......

#9 Chroogomphus

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Posted 30 March 2016 - 03:22 AM

Tasty recipe Needles,

A question:  We're forward-planning an extended trip to the mountains next autumn ( if it bloody rains), would this be a good recipe for pre-poured plates to take with me? My old glovebox would be fine to do the business in the car, but I'm wondering about a strategy to give samples as much protection as poss before I can get them home. I would have an idea of the range of fungi that I'll find so I can take batches with the nutrient side of things varied but it's the h2o2 angle or other additives that I know little about.  I'm particularly interested in a recipe that might do all this for clones of mycorrhizal species such as lactarius or hygrophorus to see if I can brew up some slurries!

Take care and good luck


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#10 Needles

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Posted 30 March 2016 - 11:43 AM

Tasty recipe Needles,
A question:  We're forward-planning an extended trip to the mountains next autumn ( if it bloody rains), would this be a good recipe for pre-poured plates to take with me? My old glovebox would be fine to do the business in the car, but I'm wondering about a strategy to give samples as much protection as poss before I can get them home. I would have an idea of the range of fungi that I'll find so I can take batches with the nutrient side of things varied but it's the h2o2 angle or other additives that I know little about.  I'm particularly interested in a recipe that might do all this for clones of mycorrhizal species such as lactarius or hygrophorus to see if I can brew up some slurries!
Take care and good luck

you should be able to take tissue samples from wild mushrooms and get good results. Just be as clean as you can. With any cloning or isolation it's good practice to be careful not to let your sample touch more than one spot on your plate. I know I have had my samples get stuck on my scalpel while making transfers or the plate gets jarred and the piece of mushroom goes rolling. I find it best to isolate away from a clone as soon as there is a enough new growth to Do so.
Best of luck on your mushroom hunt and making your clones. ...
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#11 Needles

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Posted 04 April 2016 - 01:17 PM

Today the Weilli was recovered after being in one dish of H202 agar and being transferred to a plate of mya and then to a plate of pda. It grew out very airial and was very healthy lookingimage.jpeg I harvested the mycelium from the petri plate and blended it in sterial distilled water. Then using a 50ml syringe the blended mycelium was extracted and injected into storage bottles for later use. image.jpeg
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#12 Metalneck

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Posted 26 February 2017 - 01:32 PM

Great stuff.

I have done similar but using a liquid malt culture.

 

My idea perhaps I could develop a strain more resistant to green mould. (I live in an old stone house and it's prone to green mould in the winter).

 

I transferred some 'clean' mycelium from a contaminated cake into liquid culture. Allowed the next cake to become colonised, then let it get contaminated and repeat.

 

I'm now onto generation 6 of the original comtaminated myc. The strain seems to colonise & fruit well and creat those 'cakes that will not die'.It does seem to be a resilient strain....but I'm not discounting pure luck and perhaps better aseptic technique!

 

I suppose in nature, natural selection would result in the same process but over a longer time period. Contaminated myc would have ledd chance of producing spores, resistant strains more chance.


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#13 Arathu

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Posted 10 March 2017 - 07:04 PM

Good stuff Needles...........taking notes brother!

 

A


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#14 Metalneck

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Posted 17 March 2017 - 11:54 AM

Those cakes.

Well I thought they were all flushed out a couple of weeks back. Stuck them in a carrier bag and tied it up meaning to throw them in the bin.

 

And 2 days ago I thought, "what's in that carrier bag under my desk?"

 

Lo & behold, they'd only gone and flushed inside the carrier bag again LOL.

Dunked them last night and stuck in my basic fruiting chamber.

These cakes are immortal!

 

Must remember to clone from a good specimen!






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