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InvisibleDCart
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Transferring good mycelium out of contaminated Petri dish
    #18748400 - 08/23/13 06:30 PM (10 years, 5 months ago)

Hey guys,

This is oyster mycelium that I need to transfer to a clean dish.

I was careless when putting the mycelium from the wild oyster shroom into the agar, and brought trich with it.

So my question is- if I open this dish to transfer the good mycelium to a new dish, will my glove box be ruined with these evil mold spores?



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Edited by DCart (08/23/13 06:44 PM)


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OfflineKizzle
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Re: Transferring good mycelium out of contaminated Petri dish [Re: DCart]
    #18748450 - 08/23/13 06:46 PM (10 years, 5 months ago)

The box will be fine, I assume you'll disinfecting it. You might have trouble getting a clean culture from it though. After the transfer I would do another transfer from the new culture as soon as you see some mycelium start to grow in case any mold spores got transferred along. That way you can transfer the clean mycelium away before any mold spores are able germinate, grow, and start spreading spores in the new culture.


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InvisibleDCart
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Re: Transferring good mycelium out of contaminated Petri dish [Re: Kizzle]
    #18748474 - 08/23/13 06:53 PM (10 years, 5 months ago)

Thanks Kizzle. I'll be disinfecting with 91% isopropyl and Lysol.

I'll do, at minimum, 2 transfers for sure.

If I end up losing it, I do have two other dishes that aren't showing contamination.... Yet :smile:

They were picked from a tree that grows oysters behind my house.


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InvisibleDCart
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Re: Transferring good mycelium out of contaminated Petri dish [Re: DCart]
    #18748483 - 08/23/13 06:56 PM (10 years, 5 months ago)

I just made a new batch of agar- pressure cooked at 15psi for 1 hour. Once the newly made dishes cool off, I'll make the transfer.


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OfflineKizzle
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Re: Transferring good mycelium out of contaminated Petri dish [Re: DCart]
    #18748515 - 08/23/13 07:05 PM (10 years, 5 months ago)

Bleach is probably the best disinfectant to use all around. Especially when dealing with boxes though I would just use bleach since alcohol is flammable and volatile.


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Offlinerew736
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Re: Transferring good mycelium out of contaminated Petri dish [Re: DCart]
    #18748553 - 08/23/13 07:13 PM (10 years, 5 months ago)

Agar is pretty great. If a plate has trich, just transfer it out onto a new plate. Sometimes it takes a few tries, but you can always sterilize the glovebox with alcohol, but peroxide or bleach would probably be better. Alcohol is good to kill bacteria, but spores are usually resistant. Peroxide kills spores on contact, but mycelium can create peroxidases which break down peroxide. Also an hour is WAY too long. 121deg@15psi for 15 minutes is enough. Check out fastfreds media cookbook. What medium are you using? DFA, MEA, PDA?


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InvisibleDCart
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Re: Transferring good mycelium out of contaminated Petri dish [Re: rew736]
    #18748573 - 08/23/13 07:18 PM (10 years, 5 months ago)

I am using the "grocery store agar tek" (1/4 cup agar, 2 table spoons instant potatoe, 1 teaspoon honey, 1 cup water)

I forget who made the thread. It was easy enough and one of the first few results to pop up when I was searching for agar tek. I try to use the search engine as much as possible, but I feel like it can be a crap shoot sometimes.

Do you guys have the "ultimate" agar recipe that I could look up? A link would be great, or feel free to post the recipe here. It may be useful for someone.

Thanks for your time!

O yeah, should I dilute the bleach or peroxide? How long after cleaning should I wait before working in the box with either of those?


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Edited by DCart (08/23/13 07:19 PM)


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InvisibleDCart
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Re: Transferring good mycelium out of contaminated Petri dish [Re: DCart]
    #18748587 - 08/23/13 07:22 PM (10 years, 5 months ago)

This is my first attempt at cultivation, forgive the ignorance :smile:


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OfflineKizzle
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Re: Transferring good mycelium out of contaminated Petri dish [Re: DCart]
    #18748640 - 08/23/13 07:32 PM (10 years, 5 months ago)

In a still air box mist it with a 10% bleach solution and let sit like that for 10 minutes. The falling droplets in the air help trap any particles, sort of scrubs the air clean. Then wipe the top dry to prevent dripping and use it wet. Although there's a boundry layer of still air on the surfaces in a dry box that will prevent contaminants from becoming easily airborne once they settle, the wet layer actually traps them so even more significant air movement inside the box isn't likely to stir them up.

Alternatively even soapy water works well. It's not necessary to actually kill contaminants to trap and remove them.


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Offlinerew736
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Re: Transferring good mycelium out of contaminated Petri dish [Re: Kizzle]
    #18748771 - 08/23/13 08:04 PM (10 years, 5 months ago)

Here is a agar receipe book that covers just about everything. I personally use MEA (malt extract agar), but there are tons of other ones...

http://www.shroomery.org/forums/showflat.php?Cat=&Board=59&Number=3077006&fpart=1&PHPSESSID=

As to the SAB, like Kizzle said that you have to trap the contams (but killing them is ideal). A still air box works on the principle that if the air is not moving (that much), then  neither are contam spores.

Peroxide you can use the 3% solution you buy at CVS/WalMart/Target/etc... Bleach can be used again like Kizzle said 10%. Please note: Use fresh bleach whenever possible. Sodium hypochlorite (bleach) will lose 50% of it's killing abilities in 90 days even if unopened.


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malicom said:
"Banana's emit radiation. In fact banannas are the standard in detecting solar radiation. The radiation read from a bananna tells scientists how active the sun was during the growing season. Make sure to steer clear of bananas too."
seekaytea said:
"I need to stop spending all my money on plants and take up a cheaper hobby, like heroin."
Wiccan_Seeker said:
"Your knowledge on male-on-male rape and willingness to engage in fantasy regarding the matter is commendable."
eminemvs.slimshady said:
"I want to know if they are active and magic or not. And if they are not active, can i pick them before they are active and then they will become active or do they have to grow till they activate?"


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InvisibleDCart
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Re: Transferring good mycelium out of contaminated Petri dish [Re: rew736]
    #18748797 - 08/23/13 08:10 PM (10 years, 5 months ago)

Thanks for the information, fellas.

I'll take a look at that agar link and start implementing bleach and peroxide into my sterilization methods.

Wish me luck on the transfer.


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Offlinerew736
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Re: Transferring good mycelium out of contaminated Petri dish [Re: DCart]
    #18748839 - 08/23/13 08:19 PM (10 years, 5 months ago)

What are you using/plan to use to isolate your myc? I use a scalpel that I PC'd. I think you would be able to use the sandwich tek here....
http://www.shroomery.org/forums/showflat.php/Number/2894662#2894662

Keep us posted,

Cheers!


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My trade list
malicom said:
"Banana's emit radiation. In fact banannas are the standard in detecting solar radiation. The radiation read from a bananna tells scientists how active the sun was during the growing season. Make sure to steer clear of bananas too."
seekaytea said:
"I need to stop spending all my money on plants and take up a cheaper hobby, like heroin."
Wiccan_Seeker said:
"Your knowledge on male-on-male rape and willingness to engage in fantasy regarding the matter is commendable."
eminemvs.slimshady said:
"I want to know if they are active and magic or not. And if they are not active, can i pick them before they are active and then they will become active or do they have to grow till they activate?"


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InvisibleDCart
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Re: Transferring good mycelium out of contaminated Petri dish [Re: rew736]
    #18748883 - 08/23/13 08:28 PM (10 years, 5 months ago)

I have been using sterile scalpels straight from the package or a PCed scalpel.

I have been playing around with ps. caerulescens (weilii) mycelium and transfers. I made 9 successful transfers on 8/17. I'll post a few pics for fun.

Photo taken on 8/19




Here is B1-1 on 8/23(today)

Just for fun-

 

I inoculated 5 jars of agar, following the grocery store tech. 1 got a bacterial contamination, and the other 4 cultures grew out. I labeled them A, B, C, and D. I gave C and D to a friend and kept A and B for my own experimentation.

After the cultures completely colonized, I decided to try transferring the mycelium from B to 9 Petri dishes. B was a slightly slower colonizer than A, so I figured I would try my first transfers with the weaker culture- in case I fucked it up. I don't think I did, though.

They are labeled "B1-(1-9)". "B" stands for the origional culture that it came from. "1" stands for the first transfer. "1-9" stands for the new number(label) that the dish was assigned.

As long as all goes well for B1, I'll do a second transfer to attempt to isolate fast growing mycelium. Those will be labeled B2 and so on....


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Offlinerew736
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Re: Transferring good mycelium out of contaminated Petri dish [Re: DCart]
    #18748971 - 08/23/13 08:50 PM (10 years, 5 months ago)

:asianofapproval:
I like it


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My trade list
malicom said:
"Banana's emit radiation. In fact banannas are the standard in detecting solar radiation. The radiation read from a bananna tells scientists how active the sun was during the growing season. Make sure to steer clear of bananas too."
seekaytea said:
"I need to stop spending all my money on plants and take up a cheaper hobby, like heroin."
Wiccan_Seeker said:
"Your knowledge on male-on-male rape and willingness to engage in fantasy regarding the matter is commendable."
eminemvs.slimshady said:
"I want to know if they are active and magic or not. And if they are not active, can i pick them before they are active and then they will become active or do they have to grow till they activate?"


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InvisibleDCart
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Re: Transferring good mycelium out of contaminated Petri dish [Re: rew736]
    #18749018 - 08/23/13 09:02 PM (10 years, 5 months ago)

Thanks man!

Those cultures came from a wild print that I made. I've found quite a few already and made few prints for friends and myself. Since those mushrooms and prints are somewhat abundant to me, I figured that I would try my first attempt at agar with free prints :smile:.

I hear that cultivation of that species is difficult. At some point I plan on transferring it to a spawn substrate and see what happens. I am entertaining an outdoor culture as well.


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Offlinetropicalfrenzy
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Re: Transferring good mycelium out of contaminated Petri dish [Re: DCart]
    #18751997 - 08/24/13 03:18 PM (10 years, 5 months ago)

no one mentioned it, so I will. %70 is the magic number with isopropyl (or even ethanol) to kill contaminants. Something to do with cell wall permeability or such...

I used %97 for ages till I learned this, silly me.


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Offlinerew736
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Re: Transferring good mycelium out of contaminated Petri dish [Re: tropicalfrenzy]
    #18752485 - 08/24/13 05:43 PM (10 years, 5 months ago)

^^ This

I learn something new everyday... I still prefer to use vodka... I can take a quick gulp when no one is looking...:biggrin:


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My trade list
malicom said:
"Banana's emit radiation. In fact banannas are the standard in detecting solar radiation. The radiation read from a bananna tells scientists how active the sun was during the growing season. Make sure to steer clear of bananas too."
seekaytea said:
"I need to stop spending all my money on plants and take up a cheaper hobby, like heroin."
Wiccan_Seeker said:
"Your knowledge on male-on-male rape and willingness to engage in fantasy regarding the matter is commendable."
eminemvs.slimshady said:
"I want to know if they are active and magic or not. And if they are not active, can i pick them before they are active and then they will become active or do they have to grow till they activate?"


Extras: Filter Print Post Top
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