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OfflineBabo911
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Agar Failure :(
    #5674333 - 05/25/06 07:28 PM (17 years, 8 months ago)

Tryed 6 cuttings from fresh picked mushies..... The cuttings all died and the agar has this milkey white film over it now, any idea where i went wrong?


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Invisiblemonstermitch
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Re: Agar Failure :( [Re: Babo911]
    #5674345 - 05/25/06 07:32 PM (17 years, 8 months ago)

sterilization I would certainly presume.


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OfflineBabo911
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Re: Agar Failure :( [Re: monstermitch]
    #5674353 - 05/25/06 07:33 PM (17 years, 8 months ago)

It seems like the cuttings died though?


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OfflineMrMolotov
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Re: Agar Failure :( [Re: Babo911]
    #5674392 - 05/25/06 07:47 PM (17 years, 8 months ago)

maybe they werent fresh enough?


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OfflineBabo911
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Re: Agar Failure :( [Re: MrMolotov]
    #5674401 - 05/25/06 07:51 PM (17 years, 8 months ago)

i Picked them and put them on the agar right after the veil fell.... I think i may have brused them in the process....

Next time shoudl i take a piece of mycelum near the base of the stem and would that give me a cone?


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OfflineBabo911
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Re: Agar Failure :( [Re: Babo911]
    #5674448 - 05/25/06 08:05 PM (17 years, 8 months ago)

Oh I also had it im my TiT if that metters


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OfflineMrMolotov
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Re: Agar Failure :( [Re: Babo911]
    #5674477 - 05/25/06 08:11 PM (17 years, 8 months ago)

i think your supposed to take a piece of flesh from the inside immediately after cutting the mush and placing it inside of the agar petri-dish


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OfflineBabo911
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Re: Agar Failure :( [Re: MrMolotov]
    #5674491 - 05/25/06 08:15 PM (17 years, 8 months ago)

That is what I thought I did.....


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OfflineBabo911
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Re: Agar Failure :( [Re: Babo911]
    #5674506 - 05/25/06 08:17 PM (17 years, 8 months ago)

I think I am going to try the mycelium from the caseing from where good fruit bodies were


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Invisibleshymanta
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Re: Agar Failure :( [Re: Babo911]
    #5674729 - 05/25/06 09:24 PM (17 years, 8 months ago)

A piece from the stem never worked for me. I always take a clone from inside the cap. Incubate at 80F for a day to get it going and let it continue at room temp.


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OfflineRogerRabbitM
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Re: Agar Failure :( [Re: shymanta]
    #5674807 - 05/25/06 09:45 PM (17 years, 8 months ago)

I prefer the stem. Never cut into it or your knife will push contaminants from the surface into the interior. Hold the stem with both hands at one end and tear it lengthwise down the center.

Use a flame sterilized scalpel or exacto knife to cut a very small chunk no larger than a grain of rice from the freshly exposed interior and transfer that to the petri dish. Make sure no part of the transfer is from the exterior of the fruit.

The transfer will 'look' dead for a few days, then turn fuzzy and start to grow.

A milky appearance is bacteria. Be sure to wear gloves on your hands and a surgical mask.
RR


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OfflineBabo911
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Re: Agar Failure :( [Re: RogerRabbit]
    #5674835 - 05/25/06 09:52 PM (17 years, 8 months ago)

inside like were the spores are? or cut that away? Thanks for the info, It will help much :smile:


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OfflineBabo911
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Re: Agar Failure :( [Re: RogerRabbit]
    #5674855 - 05/25/06 09:57 PM (17 years, 8 months ago)

Quote:

RogerRabbit said:
I prefer the stem. Never cut into it or your knife will push contaminants from the surface into the interior.  Hold the stem with both hands at one end and tear it lengthwise down the center.

Use a flame sterilized scalpel or exacto knife to cut a very small chunk no larger than a grain of rice from the freshly exposed interior and transfer that to the petri dish.  Make sure no part of the transfer is from the exterior of the fruit.

The transfer will 'look' dead for a few days, then turn fuzzy and start to grow.

A milky appearance is bacteria.  Be sure to wear gloves on your hands and a surgical mask.
RR




yea they all turned brown and slimey..... thanks RR so after i take it from the caseing spit it into 2 down the middle and tale a grain size, got it.

Now if i bruse it will it still be useable? I am looking for all white no blue?

I have a still air glove box so sergical mask would help from caseing to box. Also i have sergical scaples that i get for 100 for $10 (just the replacement heads) they come pre-sterial so i think i am good with that. Will try again tomorrow :smile:


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OfflineRogerRabbitM
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Re: Agar Failure :( [Re: Babo911]
    #5674904 - 05/25/06 10:09 PM (17 years, 8 months ago)

Be sure to work fast once you rip the stem. Another easy way instead of cutting a chunk is to gently scrape the scalpel blade down the inside of the freshly ripped stem and you'll cause a small 'curly' to roll up. Transfer that. Be sure to flame sterilize the blade even though it just came out of the package. Don't forget to wrap the dish with parafilm after inoculation.
RR


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semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
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OfflineBabo911
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Re: Agar Failure :( [Re: RogerRabbit]
    #5674910 - 05/25/06 10:12 PM (17 years, 8 months ago)

erf, don't have parafilm... how about eletrical tape? I think RoadKill said that would work?

Anything else that could work in a pinch?


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OfflineBabo911
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Re: Agar Failure :( [Re: Babo911]
    #5675005 - 05/25/06 10:32 PM (17 years, 8 months ago)

Or is there a place that i can get parafilm from locally?


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OfflineRogerRabbitM
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Re: Agar Failure :( [Re: Babo911]
    #5676074 - 05/26/06 06:21 AM (17 years, 8 months ago)



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semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
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InvisibleFooMan
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Re: Agar Failure :( [Re: Babo911]
    #5676095 - 05/26/06 06:44 AM (17 years, 8 months ago)

Be careful flame sterilizing your tools if you used Lysol or anything else that may be flammable inside your glovebox.


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OfflineRogerRabbitM
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Re: Agar Failure :( [Re: FooMan]
    #5676297 - 05/26/06 08:41 AM (17 years, 8 months ago)

Yea. I usually don't mention that because I'm on record saying there is no reason whatsoever to use lysol or anything else besides soap and water in a glovebox. Lysol is a surface disinfectant and you don't dump your agar or clones on the surface of the glovebox anyway. Just wipe the inside down with soap and water, or even just plain water, then put the lid on with all your stuff inside, wait five minutes for the contaminants that might be in there to settle to the bottom and go to work. It helps to mist the bottom a bit so any contaminant spores that land on the floor of the glovebox stay there.
RR


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semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
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OfflineOatman2000
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Re: Agar Failure :( [Re: RogerRabbit]
    #5677490 - 05/26/06 03:31 PM (17 years, 8 months ago)

mine failed too...I made 9 plates. I took a little chunk out of the center of the fruit, and no colonization.


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