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Offlinebjanuszewski
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trouble with my agar cloning
    #19092939 - 11/05/13 10:22 PM (10 years, 2 months ago)

tried cloning some blue oyster tissue (store bought) i did the work in a glove box and believe my sterile procedure are good as ive done pf tek grows and sawdust/straw bag grows with success (no contams)

maybe since they where store bought the tissue was to old or dry. i have a few pics to show:


pretty disgusting to look at, had to through em out. good thing i believe is there wasnt to much green. ive also tried using LC before as well and had similar results but was able to get 1 good dish with some nice looking mycelium.

kinda frustrating. agar is the way to go, very fast colonization to grain :smile: hope i can get some better success in the future.

forgot to add that i used the stems and cut them in half for fresh tissue (untouched and unexposed) ive done similar work with tissue to cardboard this way and have had success, with agar not so much :frown:


Edited by bjanuszewski (11/05/13 10:25 PM)


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OfflineRogerRabbitM
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Re: trouble with my agar cloning [Re: bjanuszewski]
    #19094062 - 11/06/13 05:07 AM (10 years, 2 months ago)

Try antibiotic agar next time.

That's bacteria.  Some is natural, but a lot of it is from all the people handling the mushrooms in the store and before.

Even if you had succeeded, you're not going to get a good commercial-worthy strain from a grocery store clone.
RR


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Offlinedeadmandave
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Re: trouble with my agar cloning [Re: RogerRabbit]
    #19094273 - 11/06/13 07:47 AM (10 years, 2 months ago)

yeah that's lots of bacteria. Another tip is to use a smaller sample size when you are cloning. i tear the mushroom in half to expose tissue that hasnt been seen or touched by anything at all, then i pick out something the size of a grain of rice or smaller with a scalpel.


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Offlinebjanuszewski
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Re: trouble with my agar cloning [Re: deadmandave]
    #19094840 - 11/06/13 11:01 AM (10 years, 2 months ago)

ill have to look into the antibiotic agar. would dipping the tissue into a peroxide solution help remove bacteria? would the tissue have to dry before touching the agar after doing that?


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Offlinethiotimoline
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Re: trouble with my agar cloning [Re: bjanuszewski]
    #19095344 - 11/06/13 01:13 PM (10 years, 2 months ago)

A peroxide dip is damaging to the mycelium as well as to bacteria, and the couple of times I tried this technique it failed horribly, so I don't recommend this.

Another method of fighting bacteria is the "hot pour-over" technique, in which you place the sample on a thin layer of agar and then pour another layer of agar on top of it. The mycelium is better than the bacteria at climbing through to the air, and when it surfaces you can transfer it to a clean plate.

If you're having more luck on cardboard (a less nutritious medium), it might also help to reduce the nutrient content of your agar mix to make it a bit less hospitable to bacteria. I haven't tried this myself though.


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Offlinedrake89
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Re: trouble with my agar cloning [Re: thiotimoline]
    #19095671 - 11/06/13 02:28 PM (10 years, 2 months ago)

it may be worth your while to get a $20 syringe of vigorous liquid culture.  If you want to clone wild mushrooms, which i find rewarding, you can take a chunk of tissue near the base.  Try to get as much as you can.  Spray it with alcohol and light it on fire, obviously not in your hand.  Then do your cloning procedure.


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Offlinebjanuszewski
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Re: trouble with my agar cloning [Re: drake89]
    #19096011 - 11/06/13 03:36 PM (10 years, 2 months ago)

ive got some dishes with yellow oyster tissue on them, which where grown from LC then spawned to sawdust. hoping these turn out better as they where obviously more fresh. will post pics later


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Offlinedrake89
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Re: trouble with my agar cloning [Re: bjanuszewski]
    #19096438 - 11/06/13 05:06 PM (10 years, 2 months ago)

ideally you would go from LC-->agar and save some plates as 'master' cultures.  but good luck!


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Offlinebjanuszewski
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Re: trouble with my agar cloning [Re: drake89]
    #19096448 - 11/06/13 05:08 PM (10 years, 2 months ago)

ok, I always thought LC wasn't good for agar. have read that its preferred to use spore syringe. but im all down for LC as I have a few jars to use up.


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Offlinethiotimoline
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Re: trouble with my agar cloning [Re: bjanuszewski]
    #19097659 - 11/06/13 08:46 PM (10 years, 2 months ago)

The trouble with liquid culture is that, unlike agar, it's not visually obvious whether your culture is clean. I do not recommend using liquid culture until you are experienced with sterile technique.


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OfflineRogerRabbitM
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Re: trouble with my agar cloning [Re: thiotimoline]
    #19098976 - 11/07/13 04:15 AM (10 years, 2 months ago)

I'm pretty experienced with sterile technique and I wouldn't ever consider making an LC.
RR


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OfflinePussyFart
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Re: trouble with my agar cloning [Re: drake89] * 1
    #19099586 - 11/07/13 09:52 AM (10 years, 2 months ago)

Quote:

drake89 said:
If you want to clone wild mushrooms, which i find rewarding, you can take a chunk of tissue near the base.  Try to get as much as you can.  Spray it with alcohol and light it on fire, obviously not in your hand.  Then do your cloning procedure.



:smbfacepalm:


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InvisibleStool_Sample
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Re: trouble with my agar cloning [Re: PussyFart]
    #19123050 - 11/11/13 08:21 PM (10 years, 2 months ago)

ooorrr... just tear down the middle and take an inner piece. ?.
One that hasn't been exposed to the air or elements outside your flowhood or SAB or GB


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Offlinebjanuszewski
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Re: trouble with my agar cloning [Re: Stool_Sample]
    #19123871 - 11/11/13 10:37 PM (10 years, 2 months ago)

here's my other agar work. this is a yellow tissue strain, looks a bit better.



looks like one got black mold :sad:, does yellow mycelium usually be more on the end of fluffy? i may have to isolate some of this i suppose?


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Offlinedrake89
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Re: trouble with my agar cloning [Re: PussyFart]
    #19128029 - 11/12/13 06:43 PM (10 years, 2 months ago)

Quote:

Notahacker420 said:
Quote:

drake89 said:
If you want to clone wild mushrooms, which i find rewarding, you can take a chunk of tissue near the base.  Try to get as much as you can.  Spray it with alcohol and light it on fire, obviously not in your hand.  Then do your cloning procedure.



:smbfacepalm:




I got this method from a professional mycologist, gary strobel.  so kindly bugger off.  the flame does not harm the tissue on the interior, and kills contams on the outside.


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OfflinePussyFart
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Re: trouble with my agar cloning [Re: drake89]
    #19128121 - 11/12/13 06:58 PM (10 years, 2 months ago)

The flame is not what worries me, it's the dipping in alcohol.

None of this is necessary, and is just added work, with no benefit.

Just put the issue culture on agar and be done with it.

If you do this in a SAB, and take from the inside of the stipe, it is already pretty clean.

There is no reason to clean the outside of the fruit.


Edited by PussyFart (11/12/13 06:59 PM)


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Offlinedrake89
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Re: trouble with my agar cloning [Re: PussyFart]
    #19128361 - 11/12/13 07:54 PM (10 years, 2 months ago)

Quote:

Notahacker420 said:
The flame is not what worries me, it's the dipping in alcohol.

None of this is necessary, and is just added work, with no benefit.

Just put the issue culture on agar and be done with it.

If you do this in a SAB, and take from the inside of the stipe, it is already pretty clean.

There is no reason to clean the outside of the fruit.




spray, not dip.  there is plenty of reason if you are in front of a flowhood.


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OfflinePussyFart
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Re: trouble with my agar cloning [Re: drake89]
    #19128368 - 11/12/13 07:56 PM (10 years, 2 months ago)

Even if you are in front of a flow hood...you rip the mushroom in half and the only air that hits the tissue is sterile.

Everybody on here has success without doing this step, so I guess to each his own.


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