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OfflineMycoAu
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Tissue culture from small mushrooms
    #8010751 - 02/11/08 10:26 PM (6 years, 7 months ago)

What's the best way to isolate clean tissue from a small mushroom? (total height less than 3cm and thin caps, thin stalks)

Taking suggestions- trying to do it by hand has rendered badly contaminated agar cultures (bacteria from hands).


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Invisibled4a2n0k
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Re: Tissue culture from small mushrooms [Re: MycoAu]
    #8010875 - 02/11/08 10:53 PM (6 years, 7 months ago)

I have had good luck cloning small pins on agar. To do so I would put a tray in front of a flowhood and pick with sterile tweezers.

If your going with a glovebox, I would use a lager fruit. That way you can split the stem and take an unexposed chunk from the middle. You don't need anything huge, about the size of a grain of rice.


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OfflineMycoAu
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Re: Tissue culture from small mushrooms [Re: d4a2n0k]
    #8018003 - 02/13/08 04:47 PM (6 years, 6 months ago)

Sorry, I read my post and realize I wasn't being clear. I want to know how to manipulate a small mushroom to remove that peice of tissue without contiminating it in the process. I don't have problems with hand dexterity or will aseptic technique, so anything you want to suggest is probably well within my capabilities. Just looking to find a possibility for an easier method with lower chances of contamination from touch.


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Offlinecrazyeddie
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Re: Tissue culture from small mushrooms [Re: MycoAu]
    #8018035 - 02/13/08 04:56 PM (6 years, 6 months ago)

tweezers and a scalpel. don't forget to sterilize them.


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Invisibleuncle_rico
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Re: Tissue culture from small mushrooms [Re: MycoAu]
    #8018048 - 02/13/08 04:58 PM (6 years, 6 months ago)

would 'core biopsy' work for you?

If you are going to agar, the liquid used to shoot the core biopsy out of the needle would be problematic.

maybe .... shoot the tissue sample into H2o2, then recover with tweezers to agar.


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OfflineCiv
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Re: Tissue culture from small mushrooms [Re: uncle_rico]
    #8018063 - 02/13/08 05:02 PM (6 years, 6 months ago)

Give it a h202 dunk, put it in a blender with h20/h202, make a myco shake/slurry... use to inoculate medium of your choice.

Run around like jonny apple seed pouring it into peoples potted plants.


--------------------
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So, wash it well afterwards & DON'T tell them..."  -Agar


Edited by Civ (02/13/08 05:03 PM)


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Re: Tissue culture from small mushrooms [Re: MycoAu]
    #8018075 - 02/13/08 05:05 PM (6 years, 6 months ago)

Try THIS


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:rules:


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OfflineMycoAu
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Re: Tissue culture from small mushrooms [Re: FooMan]
    #8024541 - 02/14/08 11:08 PM (6 years, 6 months ago)

Fooman, that write-up is amazing- but I've already read it. I wish all teks/methods were so clearly written. I think that the core biopsy might work, but I'm not sure. The stalk might not be large enough. Plus, that method still requires external material to be included in the culture (greater potential for contamination).

I almost always soak tissues in 3% H2O2 for 5-15minutes before plating or adding to LC. This is the first time I've had a contamination issue like this (usually in the 1-5% contam rate with non-sterile starting materials). My last run was 100% contaminated which makes me think that my peroxide was contaminated or that I was unaware of touching the tissue being removed. I'm sure that everything else was just fine.

The next time I obtain a sample of that species to work with, I'll try a fresh bottle of peroxide along with a few plates that try the biopsy method.

Thanks for the input.

And btw, does anybody have experience with germinating shitake spores? I'm currently waiting on some plates to germinate (been one week- no germination or contamination). I want to know if pre-hydration in sterile water is recommended or necessary, if LC multispore is a good place to start in place of starting directly on agar and how long they typically take to germinate under a partcular set of conditions.

Thanks again for the help!


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OfflineRogerRabbitM
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Re: Tissue culture from small mushrooms [Re: MycoAu]
    #8024763 - 02/14/08 11:54 PM (6 years, 6 months ago)

You don't have to do anything if you have freshly poured petri dishes and small pins. You can simply pluck the pins and place them right on the agar. Within a couple of days, mycelium will be growing out well away from the fruits and any contaminants that were on them, will be left behind. Take a small pieces of mycelium from the leading edge after four or five days, and transfer them to a new petri dishes. Result-clean mycelium with little sterile technique involved other than when you pour the agar.

It's important if using this cloning tek to use rapidly growing pins so that the mycelium will outrun any contaminants. It won't work well with old, or slow growing fruits. The pictures below were only taken a few days apart.
RR


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OfflineMycoAu
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Re: Tissue culture from small mushrooms [Re: RogerRabbit]
    #8027794 - 02/15/08 05:25 PM (6 years, 6 months ago)

Wonderful information to know. However, I'm not working with pins- only wild fruits found in a local park. Once I've managed to fruit a culture that I've been successful in cultivating, I will try the pin/agar method to expand my mycelial bank of that strain.


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InvisibleSmushroom
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Re: Tissue culture from small mushrooms [Re: MycoAu]
    #8028506 - 02/15/08 08:12 PM (6 years, 6 months ago)

I am about to do an LC using small pins out of necessity as well since I haven't started doing agar work yet. Here is what I am planning on trying and I added a few things since you are picking them in the wild and not in a controlled environment.

I am going to do it in a still air box inside a homemade clean room. I would have to say the less air movement you have the better (unless you have a flow hood).

Get everything you will need together:

For Harvest
Aluminum foil, preferably heavy duty
Tweezers
Butane Lighter
Hand sanitizer
Alcohol

For Extraction
Alcohol (preferably the 91% stuff)
Butane lighter or alcohol lamp (I use a metal shot glass with alcohol in it)
Empty Syringe (PC is possible, boil if not)
Scalpel or new exacto knife
Paper towels
Agar dish
Hand sanitizer

Harvesting
Before you go to harvest take a piece of foil and wipe with alcohol, fold in half and fold the edges up.
Wipe tweezers with alcohol and wrap in alcoholed foil
Take the foil, tweezers, ligher, hand sanitizer to where you will harvest.
Find the shroom.
Sanitize your hands, open the tweezers and flame them with the lighter.
Pick the shroom.
Open the alcoholed foil and put inside and fold back up.

Cloning
PC or boil the syringe and wrap in alcohol foil.
Same with scalpel/exacto.
Put everything in the work area and spray with lysol.
Open the foil with the shroom.
Take out the scalpel/exacto and flame it.
Cut of the base of the shroom.
Take out the syringe, flame the needle, draw in a small amount of air.
Stick the syringe as far up the cut end of the stem as you can, don't let it puncture the stem wall.
Expel the syringe onto the agar dish, should yield a small amount of myc even if it isn't very visible.
Let that grow out.


That seems to be the most sanitary method of getting clean tissue out of the shroom. Let me know if you try it and it works.


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InvisiblePeterthinks
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Re: Tissue culture from small mushrooms [Re: Smushroom]
    #8029482 - 02/16/08 12:22 AM (6 years, 6 months ago)

You just need a really steady hand and some tape.
Tape the mushroom down to something sterile, make a shallow cut in the stem and peel it up...you just want to expose the inner flesh, don't touch the knife to the inner stuff while peeling, use tweezers if you have to.
heat the knife cherry hot and let it cool, scrape the tip of the knife up and down till a small piece is loose, don't let it touch the outside of the mushroom.
tip it into your agar plate or liquid culture or sterile grain or whatever.
Make a few, failure rate is understandably high.
I didn't think I could do it either but I cloned a grocery store  enokie once like this. they got caps smaller than thumbtacks and 1 to 2/16th inch thick stems.
here's a pic of the enokie clone fruiting in it's 150mm plate.

they just get longer...not much thicker (when grown in high CO2 like the store ones)
:jester:


--------------------
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Set a man on fire and he will be warm for the rest of his life.
NEWB NEWBIE NEWCOMER IGNORANT? QUESTIONS?
Click HERE HERE HERE HERE For detailed instructions with pictures on how to grow mushrooms. There is a lot of info on the Shroomery and this is what you need to know.


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Offlineudok
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Re: Tissue culture from small mushrooms [Re: Peterthinks]
    #8030124 - 02/16/08 06:23 AM (6 years, 6 months ago)

I buy premade MEA , PDA, SAB GC plates with chloramphenicol and gentamicin. 38 Euro for 100 plates. Until now, there was no need for a
third transfer to get rid of trichoderma or penicillium. Only to tell
you that i try my best on the first part. Not more.


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InvisiblePeterthinks
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Re: Tissue culture from small mushrooms [Re: udok]
    #8030862 - 02/16/08 01:20 PM (6 years, 6 months ago)

Just being cautious.If he's trying to clone from one wild mushroom it's better to make a bunch of plates rather than one.I don't need to do second transfers to get rid of contams cause if I do say 10 plates at least one will be clean.(if there are more clean ones you can trade them anyway)I only transfer to isolate sectors.
But I make my own PDA agar for like $0.02 a plate so it doesn't matter how many I screw up.
You never know when or if you'll run into that wild mushroom again so you really have to stack the odds in your favor.
:jester:


--------------------
Give a man a fire and he will be warm for the rest of the night.
Set a man on fire and he will be warm for the rest of his life.
NEWB NEWBIE NEWCOMER IGNORANT? QUESTIONS?
Click HERE HERE HERE HERE For detailed instructions with pictures on how to grow mushrooms. There is a lot of info on the Shroomery and this is what you need to know.


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InvisibleSmushroom
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Re: Tissue culture from small mushrooms [Re: Peterthinks]
    #8032014 - 02/16/08 07:53 PM (6 years, 6 months ago)

That is true. I was just thinking since the stem is thin you may only get one good biopsy out of it before you start to pierce the side of it.


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InvisiblePeterthinks
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Re: Tissue culture from small mushrooms [Re: Smushroom]
    #8032076 - 02/16/08 08:11 PM (6 years, 6 months ago)

If you can see it it's big enough. And a piece twice as big doubles the chance you brought a contaminate into the dish with the biopsy.
I'm not saying it isn't a total pain in the ass to get 6 samples out of a tiny shroom, just that it can be done. I'd avoid doing it entirely if I didn't have to clone store bought enokie once in a while.
I know they come in big bunches but as long as you got one peeled and taped down you might as well take a bunch of clones.
Spindly little bastards!
:jester:


--------------------
Give a man a fire and he will be warm for the rest of the night.
Set a man on fire and he will be warm for the rest of his life.
NEWB NEWBIE NEWCOMER IGNORANT? QUESTIONS?
Click HERE HERE HERE HERE For detailed instructions with pictures on how to grow mushrooms. There is a lot of info on the Shroomery and this is what you need to know.


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