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OfflineTheReckoning
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Registered: 05/13/09
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Last seen: 1 year, 11 months
Potato Dextrose Agar WITHOUT Petri Dish and Liquid Culture Question
    #10350240 - 05/16/09 08:45 PM (14 years, 10 months ago)

I started a liquid culture with 100ml of water and 4 grams of organic honey. I did a tissue transfer by pricking the stem of a mature mushroom with a gauged syringe and injecting that tissue into my liquid culture jar. The solution has a bunch of white particles floating around, which creates a sort of cloudy effect when I stir it. I started the culture 5 days ago and this is all that I have seen in the liquid culture. Does it sound like my liquid culture failed?

Another question on the topic of using Potato Dextrose Agar. I have a vial of it that has a self healing injector site cap. What I did was remove the cap and place a cut piece of mushroom tissue and placed it in the vial of PDA. Is this a correct method to start an agar culture? Thanks!


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InvisibleDoc_T
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Re: Potato Dextrose Agar WITHOUT Petri Dish and Liquid Culture Question [Re: TheReckoning]
    #10350301 - 05/16/09 09:04 PM (14 years, 10 months ago)

LC might be ok, shoot a test jar in a couple weeks.

The vial thing, hard to say without more info. It was prepared agar, not powder or something? The you should be ok, if you used good sterile technique.


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OfflineTheReckoning
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Registered: 05/13/09
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Re: Potato Dextrose Agar WITHOUT Petri Dish and Liquid Culture Question [Re: Doc_T]
    #10350332 - 05/16/09 09:13 PM (14 years, 10 months ago)

It is a vial of prepared PDA, 100% sterile. I built a glove box out of a Sterilite large clear bin, put a plastic garbage bag over the opening and taped it good with duct tape, made two holes and duct taped the edges to reinforce. I then wiped down the inside with alcohol and sprayed Lysol. I wore latex gloves and washed them with alcohol followed by hand sanitizer. Then I proceeded to do the tissue transfer. The tissue bruised before I was able to get it into the vial, so it was blue. Does that matter at all if it will work?

As for the liquid culture, I started another larger subculture, but there is a lot of floating stuff in there, and it wasn't in there before. It looks strange, maybe honey sediment? It's kind of stringy.


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A scapegoat, for all your insecurities. Excuse me; a hero; a man made god; for all your insecurities - Remembering Never


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Offlinemushroomhunter10
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Re: Potato Dextrose Agar WITHOUT Petri Dish and Liquid Culture Question [Re: Doc_T]
    #10350337 - 05/16/09 09:14 PM (14 years, 10 months ago)

It's difficult to assess whether or not your LC is contaminated or not. It's why you should have either used regular agar plates, or even transferred said culture to grains. RogerRabbit set me straight on the "dangers" of LC's.

Don't get me wrong, LC's are awesome, but for novices (I'm not implying anything.) it's difficult to discern whether or not it's mycelium or a contamination.

Agar or grains next time. :thumbup:

As far as your agar question. You did it correctly, but the environment and sterile technique could be questionable without more detail.

Good Luck


--------------------
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OfflineTheReckoning
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Registered: 05/13/09
Posts: 411
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Re: Potato Dextrose Agar WITHOUT Petri Dish and Liquid Culture Question [Re: mushroomhunter10]
    #10350349 - 05/16/09 09:17 PM (14 years, 10 months ago)

I actually am testing the master liquid culture on grains now, I inoculated yesterday. How long before I should be able to see some mycelium growth?


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A scapegoat, for all your insecurities. Excuse me; a hero; a man made god; for all your insecurities - Remembering Never


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Offlinemushroomhunter10
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Re: Potato Dextrose Agar WITHOUT Petri Dish and Liquid Culture Question [Re: TheReckoning]
    #10350381 - 05/16/09 09:25 PM (14 years, 10 months ago)

Mycelium is surprisingly fast. Two days and you should see some action. A week at most.

The bruising, it's all good. It's just psilocin's reaction to the air as it breaks down. Really, nobody really knows why it turns blue. :shrug:


May I ask which species you're attempting to clone?


--------------------
Imagine if you needed it and it wasn't there... GIVE BLOOD
Get a free (PAINLESS) bone marrow testing kit and help save lives HERE.
Jesus if you're reading this, please come back already. We need you now more than ever!
The U.S. Constitution!

Best WBS Tek
EZ Potato-Honey Agar Tek
MY TRADES

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OfflineTheReckoning
:)

Registered: 05/13/09
Posts: 411
Last seen: 1 year, 11 months
Re: Potato Dextrose Agar WITHOUT Petri Dish and Liquid Culture Question [Re: mushroomhunter10]
    #10350417 - 05/16/09 09:36 PM (14 years, 10 months ago)

P. cubensis Ecuadorian I believe. I got my specimens from my friend here in town, and performed the transfer to PDA yesterday. In the PDA I see translucent pieces of some sort of debris in various places. How long before I should see some growth in the agar?


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A scapegoat, for all your insecurities. Excuse me; a hero; a man made god; for all your insecurities - Remembering Never


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Offlinemushroomhunter10
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Re: Potato Dextrose Agar WITHOUT Petri Dish and Liquid Culture Question [Re: TheReckoning]
    #10350426 - 05/16/09 09:40 PM (14 years, 10 months ago)

If it's a tissue culture then you can see growth in as little as 48 hours, albeit small, but nonetheless growth. Spores take longer because they have to germinate. Sometimes 24 hours you can see VERY minor changes with tissue culture. The thing you'll see first are pieces of mycelium "reaching out" to the new food source.

Give it a week and you'll know if it's good to go.

Smoke a J, and relax. You'll get there. :smile:


--------------------
Imagine if you needed it and it wasn't there... GIVE BLOOD
Get a free (PAINLESS) bone marrow testing kit and help save lives HERE.
Jesus if you're reading this, please come back already. We need you now more than ever!
The U.S. Constitution!

Best WBS Tek
EZ Potato-Honey Agar Tek
MY TRADES

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OfflineTheReckoning
:)

Registered: 05/13/09
Posts: 411
Last seen: 1 year, 11 months
Re: Potato Dextrose Agar WITHOUT Petri Dish and Liquid Culture Question [Re: mushroomhunter10]
    #10350433 - 05/16/09 09:43 PM (14 years, 10 months ago)

Will do :wink: I think my first liquid culture may have failed, but without contamination, just some plain honey water. I injected this into a rye grain jar to test. If it is a failed culture, can I still use that rye grain jar to grow?


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A scapegoat, for all your insecurities. Excuse me; a hero; a man made god; for all your insecurities - Remembering Never


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Offlinemushroomhunter10
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Re: Potato Dextrose Agar WITHOUT Petri Dish and Liquid Culture Question [Re: TheReckoning]
    #10350458 - 05/16/09 09:52 PM (14 years, 10 months ago)

If the culture failed to grow, and there weren't contaminants then you can use that same jar to grow.

I would just make a new jar instead though. Less worry/headache/anxiety.

Here is the real thing to do though.

Read
Read
Read

When you're done reading, read some more! :grin:

If I was you I'd ask your friend for some spores and just knock-up some grains instead of trying three or four methods at once.


--------------------
Imagine if you needed it and it wasn't there... GIVE BLOOD
Get a free (PAINLESS) bone marrow testing kit and help save lives HERE.
Jesus if you're reading this, please come back already. We need you now more than ever!
The U.S. Constitution!

Best WBS Tek
EZ Potato-Honey Agar Tek
MY TRADES

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InvisibleDoc_T
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Re: Potato Dextrose Agar WITHOUT Petri Dish and Liquid Culture Question [Re: mushroomhunter10]
    #10350481 - 05/16/09 09:58 PM (14 years, 10 months ago)

Agreed.
You sound pretty knowledgeable and reasonably competent. Put those skills to work using a proven technique first, then experiment. But wait a week or more before you call this attempt a failure or success.


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OfflineTheReckoning
:)

Registered: 05/13/09
Posts: 411
Last seen: 1 year, 11 months
Re: Potato Dextrose Agar WITHOUT Petri Dish and Liquid Culture Question [Re: mushroomhunter10]
    #10350527 - 05/16/09 10:09 PM (14 years, 10 months ago)

I already got a spore print from a fairly large specimen last week, and used those spores to start a liquid culture the other day. That jar of liquid culture though, has a bunch of sediment floating in it, I expect it to be from the honey but I'm not sure. Here are some pics of the culture started with spores: (notice that there is a build up of white residue on the edge, it doesn't move when I swirl the jar to create a vortex)



Let me know what you think, thanks. :smile:


--------------------
A scapegoat, for all your insecurities. Excuse me; a hero; a man made god; for all your insecurities - Remembering Never


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InvisibleDoc_T
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Re: Potato Dextrose Agar WITHOUT Petri Dish and Liquid Culture Question [Re: TheReckoning]
    #10350533 - 05/16/09 10:11 PM (14 years, 10 months ago)

LC is always a gamble. You can't tell much by looking, you have to wait and shoot up a test jar.


--------------------
You make it all possible. Doesn't it feel good?

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Offlinemushroomhunter10
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Re: Potato Dextrose Agar WITHOUT Petri Dish and Liquid Culture Question [Re: Doc_T]
    #10350537 - 05/16/09 10:11 PM (14 years, 10 months ago)

Yeah and definitely watch RR's videos. He's quite a character and a super-exceptional cultivator.


--------------------
Imagine if you needed it and it wasn't there... GIVE BLOOD
Get a free (PAINLESS) bone marrow testing kit and help save lives HERE.
Jesus if you're reading this, please come back already. We need you now more than ever!
The U.S. Constitution!

Best WBS Tek
EZ Potato-Honey Agar Tek
MY TRADES

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