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InvisibleToadstool5
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Cloning Question Need Help Quickly
    #22866364 - 02/05/16 02:16 AM (8 years, 1 month ago)

I am being trusted with cloning Helvella dryophila to agar dishes for future studies and I am somewhat confused as to what would be optimal for such an unusual morphology.



As you can see the hymenium is on the outside of the cap and the stipe is very furrowed so tearing into the inner layers of flesh to expose clean tissue might not be possible. It doesn't seem as simple as, "tear it open, flame, and cut a piece!" :lol:

I plan on washing the specimen several times in sterilized tap water followed by a sterile solution of 10% bleach but I feel this might not be enough. Should I also do a very quick dunk into a 10% lugol's iodine or 10% povidone-Iodine solution?

I am also curious as to whether or not it might be more advantageous to peal away the hymenium, treat the underlying pileus with 10% lugol's or povidone-Iodine, and clone to agar? I do not know the average thickness of tissue underneath the hymenium compared to the average thickness of the stipe. I am essentially going off of pictures as I have not yet obtained the mushroom yet.

Someone with experience in cloning odd mushrooms please help! This species has not been studied very much and this work will hopefully help produce another formal collection.

On a side note: What is the best way to collect ascospores for microscopy work? Would allowing them to naturally drop into a 1.5ml centrifuge tube work or should I scrape/swab some into a certain container?

Thanks shroomery! You guys fuckin' rock :rockon:


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If you do not know where the mushroom products you are consuming are grown, think twice before eating them. :badshroom:
- Paul Stamets

AMU Teks :mushroom2: Stro's Write Ups

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InvisibleRogerSmith

Registered: 01/29/15
Posts: 365
Re: Cloning Question Need Help Quickly [Re: Toadstool5]
    #22866401 - 02/05/16 02:46 AM (8 years, 1 month ago)

Don't soak mushroom. Bacteria from outside will get easier inside

Success depends mostly on your previous experience and species you are cloning, not some knowledge that solves your problem lyk in theory.
Cloning wild mushrooms is not easy, one can fail a lot.

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InvisibleToadstool5
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Re: Cloning Question Need Help Quickly [Re: RogerSmith]
    #22866678 - 02/05/16 06:10 AM (8 years, 1 month ago)

I'm pretty sure with how thick the tissue is and it being picked from standing water it is already completely saturated with bacteria if not something even harder to isolate like spores.

I feel technique has a huge difference in success. Surgeons prep for their work so when doing the equivalent of a minor biopsy on a fruit it's probably wise to prep too. We have to know the individual (old, yound, small, large, infectious, healthy), we have to prep the individual for the operation, control your vectors while working, and finally take the biopsy and place it on media for culturing.

Not preping the fruit, seems kinda reckless seeing as how this is our only chance of culturing (we only have a single fruit) the species. We are also limited on resources so I'm not going use dilution plating techniques like i'm digging for a glomeromycetes in a pile of compost.


--------------------
If you do not know where the mushroom products you are consuming are grown, think twice before eating them. :badshroom:
- Paul Stamets

AMU Teks :mushroom2: Stro's Write Ups

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InvisibleRogerSmith

Registered: 01/29/15
Posts: 365
Re: Cloning Question Need Help Quickly [Re: Toadstool5]
    #22866708 - 02/05/16 06:36 AM (8 years, 1 month ago)

You seem to know what you are doing and there is no special knowledge really to solve such situation.
The thing that makes difference between failure and success is experience and all the learning it comes with.
If mushroom is soaked wet, I don't think you have much chances at all. Even with antibiotic agar.
You can try collecting spores but there will be much dirt between them I guess.
With wild mushrooms, you have to try and utilize everything you know but sometimes it is not enough. It doesn't get any better than this

As I mentioned in my first post, success also depends on species. I have no knowledge about your mushroom. If it is aggressive like ganoderma, pleurotus, than it is totally possible to clone soaking wet mushroom

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InvisibleToadstool5
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Registered: 01/22/15
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Re: Cloning Question Need Help Quickly [Re: RogerSmith]
    #22866729 - 02/05/16 06:52 AM (8 years, 1 month ago)

Very true, a lot of it is simply luck and what you are working with. I'm trying to increase my odds as best as i can because i don't want to let my peers down even if its futile. There is a lot of pressure when you are the person picked to keep something alive. :confused:

I wish he had found several fruits and i had an unlimited amount of agar!

I'm going look up some TEKs for transfering to hot agar. I've seen some promising results from it but I can't remember what temperature the agar should be at when you transfer the clone.


--------------------
If you do not know where the mushroom products you are consuming are grown, think twice before eating them. :badshroom:
- Paul Stamets

AMU Teks :mushroom2: Stro's Write Ups

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InvisibleRogerSmith

Registered: 01/29/15
Posts: 365
Re: Cloning Question Need Help Quickly [Re: Toadstool5]
    #22866734 - 02/05/16 06:56 AM (8 years, 1 month ago)

Hot agar works. Pretty good. It usually just stuns bacteria enough for mycelium to take over. Success and temperature question depends on species. Again. Try different temperatures. Mycelium can survive it pretty high. Above 60 C agar I mean.

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InvisibleToadstool5
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Re: Cloning Question Need Help Quickly [Re: RogerSmith]
    #22867468 - 02/05/16 12:36 PM (8 years, 1 month ago)

Thanks! That gives me a rough estimate on where to start, I've never tried it but it seems like a fun way to try and outrun bacteria.

I might do two clones on room temperature agar to get a better visibility of what is contaminating the cultures and then do the rest with hot agar since I have no gentamicin or lactic acid.

I'm also considering trying to run a very small piece on pieces of paper soaked in dilute nutrients, run a piece on cardboard, and germinate ascospores in sterilized, distilled water. I figure the other contams might have a more difficult time on more complex sources of sugar/carbon or lower nutrient concentrations. Eventually I'll be storing dry samples on paper squares in glassine bags according to Cornell University's write-up so that might even save me some time and work.

https://blog.mycology.cornell.edu/2008/01/10/a-simple-way-to-preserve-fungal-cultures/

I'm uncertain of how I will keep the samples at the correct temperatures but I believe the government has storage facilities that can store them once we turn in our work. We will have to try our best to quickly store them too! Cornell mentions a loss of pathogenicity and the group that is interested in these samples would definitely want to know if the local ascomycetes are pathogenic towards the local plants. Our connection to the group is through a botanist and the manager of the land so they don't understand much about fungi other than it can destroy a lot of plants very quickly :lol:

They are also located in California Chaparral and Helvella dryophila supposedly forms EcM connections with several Oak specieis, we don't really understand the implications of this bond and if it also forms arbuscular connections. Many EcM species are being uncovered as AM species with better staining and imaging techniques of roots so it's definitely something to consider.

I'm so excited and nervous! I get the sample sometime today after 4:00pm PT or Sunday around midday. :willynilly:

Normally I work independently so I don't care if I mess up my projects and fail, It'll be fun to work under some extra pressure. I like a good challenge.


--------------------
If you do not know where the mushroom products you are consuming are grown, think twice before eating them. :badshroom:
- Paul Stamets

AMU Teks :mushroom2: Stro's Write Ups

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InvisibleRogerSmith

Registered: 01/29/15
Posts: 365
Re: Cloning Question Need Help Quickly [Re: Toadstool5]
    #22868268 - 02/05/16 03:50 PM (8 years, 1 month ago)

Ok so I checked what you are cloning so I'm not giving you bad advice.
Not much information about this mushroom. I assume it is mycorrhizal? Mycorrhizal species are slow on agar. I tried cloning nice healthy Boletus mushroom few months ago. 14 days nothing and than bacteria. Pathetic compared with other saprophytes.
I don't think you have much chances to isolate mycelium. Cardboard, antibiotics or anything else.

Which leaves spores.
Easy way is to use sterile swab. That way you will sample spores together with all the other microorganisms. But you will have spores.
You could try working something out under microscope and laminar but that is beyond me.

Don't take anything for granted, try everything yourself. These are just general tips

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InvisibleToadstool5
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Registered: 01/22/15
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Re: Cloning Question Need Help Quickly [Re: RogerSmith]
    #22869299 - 02/05/16 10:47 PM (8 years, 1 month ago)

Yeah I believe it's an EcM species. It doesn't actually penetrate the roots but it does form a sheath around the tips, I don't have much information on the mycorrhiza.

I did find this article regarding lactose and glucose concentrations of PDA and MEA with H. crispa:

http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.403.584&rep=rep1&type=pdf

It's probably a somewhat slow species but they seemed to have completed H. crispa within an ok time frame. My friend was also able to give me two fruits. One is missing most of the stipe but I am confident that I can collect spores from it or clone under the hymenium. Between two fruit bodies and spores I think I can get something salvageable.

Tomorrow is the big day! I'll probably post results regardless of my results so if someone else has a weird fruit body from a filthy environment they can compare notes and copy my success or avoid my failure :lol:


--------------------
If you do not know where the mushroom products you are consuming are grown, think twice before eating them. :badshroom:
- Paul Stamets

AMU Teks :mushroom2: Stro's Write Ups

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Invisibleforagedfungus
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Re: Cloning Question Need Help Quickly [Re: Toadstool5]
    #22870383 - 02/06/16 07:55 AM (8 years, 1 month ago)

My only advice is make to up as many plates as you can.
I cloned some cordyceps militaris last summer. It took over 20 clone attempts (6 diferent fruits) before I got one that grew faster than the bacteria.

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InvisibleNakedmushroomguru
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Re: Cloning Question Need Help Quickly [Re: Toadstool5]
    #22870413 - 02/06/16 08:11 AM (8 years, 1 month ago)

Most of the time when I culture im a little unordinary I dip the piece of tissue in ozonated water . Most people are scared of it but have been using  for last little bit with no problems and very low contamination rates. It takes the piece of tissue a couple days to bounce back but is my preferred method. And the white hellvela I cloned this past summer worked very well with this method . I think there is actual picks of it in the identification forum I posted before my laptop crashed.I also use antibiotic agar when culturing wild fruits

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InvisibleRogerSmith

Registered: 01/29/15
Posts: 365
Re: Cloning Question Need Help Quickly [Re: Nakedmushroomguru]
    #22870452 - 02/06/16 08:39 AM (8 years, 1 month ago)

This gave me an idea. You could put a few pieces of inner tissue in jar of water for a few weeks. It is contaminated already with bacteria so the goal here is to make mycelium stronger while bacteria doesn't grow much because there are no nutritions and the water is sterile of course. Observe it and if you see mycelium "hairs" growing from tissue (watch under good light) you can be sure it is mushroom mycelium. Than it will be much easier to isolate it on agar. But you will also have bacteria in this water. Still better chances sometimes than going straight to agar. Do both of course.
It helps draining excessive moisture from tissue with sterilized towel so the wet spot doesn't promote bacteria.
This is very helpful because mycelium sometimes need to rehydrate. I wouldn't wait more than 30 days. Make sure jar has gas exchange.
If this doesn't bring mycelium back to life nothing will.
I tried this method a few times and it works.

More dishes&samples, more chances of success yes :thumbup:

Edited by RogerSmith (02/06/16 08:48 AM)

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InvisibleRaven44
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Re: Cloning Question Need Help Quickly [Re: RogerSmith]
    #22870463 - 02/06/16 08:47 AM (8 years, 1 month ago)

A method tradd cotter uses for wild clones is to allow the fruit bodies to dehydrate by air drying. For a controlled amount of time which is dependent on the specimen at hand.

I have used this method to clone turkey tails and ps cyans from the wild w success.

I allowed the specimens to dry for a day or two in a paper bag.

They were quite dry maybe 50 percent dry I might dare say.

I have both of these on agar now from wild clones. Works great and I'd highly reccomend it.

So I personally wouldn't dunk it. I'd dry it out to get rid of the bacteria...

I always put 2-4 samples on one dish also so I'd just attack that fruit. And then let us know what area took best... :smile:

Good luck

I think everyone having problems should do this. And also I have found using your old month old dishes is best cause the agar surface is nice and dry and doesn't seem to support bacteria as much.

First try, four samples each total. Might have only been two samples each.

So I took two dishes and out two cy samples on each one. Got a nice rhizomorphic growth from one sample. W the turkeys I got two nice samples.

Honestly I think I only took two samples of each. So one dish of Cy and one dish od turkey tails, two samples on each dish. 1/2 cy samples took. 2/2 turkey tail samples took.

I repeated the process w new fruits and new dishes. (Not month old dishes. ) no samples took. I have noticed new dishes not working (contaminating) and old dishes working in past store bought clone experiments also

Edited by Raven44 (02/06/16 08:54 AM)

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InvisibleRogerSmith

Registered: 01/29/15
Posts: 365
Re: Cloning Question Need Help Quickly [Re: Raven44]
    #22870474 - 02/06/16 08:52 AM (8 years, 1 month ago)

Turkey tail and P.Cyan are saprophites. Trametes is very agressive, idk about P.Cyan, but mycorrhizal are very weak.
So the point in my suggestion is not because of issue with bacteria. The point is to give mycelium chance to win against bacteria.

I would try all if I were op. I wouldn't limit myself to just few methods if I really wanted to get the culture.

As I said, the more, the better.

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InvisibleRaven44
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Re: Cloning Question Need Help Quickly [Re: RogerSmith]
    #22870487 - 02/06/16 08:58 AM (8 years, 1 month ago)

Quote:

RogerSmith said:
Turkey tail and P.Cyan are saprophites. Trametes is very agressive, idk about P.Cyan, but mycorrhizal are very weak.
So the point in my suggestion is not because of issue with bacteria. The point is to give mycelium chance to win against bacteria.

I would try all if I were op. I wouldn't limit myself to just few methods if I really wanted to get the culture.

As I said, the more, the better.




Rock on, I didn't know this makes sense. This may help a lot still tho :smile:



Turkeys



Cys

Bacteria growth was very very slow, got taken over

I have zero experience w mychorizal species cloning. Hope it goes well :smile:

Edited by Raven44 (02/06/16 09:23 AM)

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