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OfflineMycoWill
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Is this bacteria on this petri dish? * 1
    #26637821 - 04/29/20 10:35 PM (3 years, 8 months ago)




I attempted to clone 5 different mushrooms from a MS tub recently and every one of my 15 petri dishes looks likes these. Are these all contaminated with a bacterial infection? And is it still possible to obtain a clean sample as the mycelium grows out?


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OfflineLogicaL ChaosM
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Re: Is this bacteria on this petri dish? [Re: MycoWill]
    #26637823 - 04/29/20 10:37 PM (3 years, 8 months ago)

Looks like bacteria and mycelium to me. The bacteria is the slime and the white fuzzy stuff is the mycelium.


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Re: Is this bacteria on this petri dish? [Re: LogicaL Chaos] * 1
    #26637832 - 04/29/20 10:41 PM (3 years, 8 months ago)

yup


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Re: Is this bacteria on this petri dish? [Re: tripdawg420]
    #26637837 - 04/29/20 10:43 PM (3 years, 8 months ago)

:solidnod:


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OfflineJe77Ce11ar
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Re: Is this bacteria on this petri dish? [Re: LogicaL Chaos] * 2
    #26637863 - 04/29/20 11:05 PM (3 years, 8 months ago)

Def bacterial. Just wait til the myc grows out and try to take a little piece and transfer a cpl times you should be good. That or you could use a scalpel/inoculation loop to try and scrape off some myc growth and transfer to new plate


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Re: Is this bacteria on this petri dish? [Re: Je77Ce11ar]
    #26637867 - 04/29/20 11:07 PM (3 years, 8 months ago)

That is some Top-Shelf Advice! :solidnod:


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OfflineMycoWill
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Re: Is this bacteria on this petri dish? [Re: LogicaL Chaos] * 1
    #26637870 - 04/29/20 11:11 PM (3 years, 8 months ago)

Thanks bks a ton for the quick advice, I will transfer it out a few times until I get a clean dish. Any tips on procedures to reduce this in the future? Would the bacteria have been more likely on the mushroom or introduced in the lab process? I left 3 dishes alone and they are still in front of my flow hood with no bacteria at all.

I have been have bacteria issues all around since day 1. However the plates that inoculated these grains were clean. Besides proper fae, is there anything else I can do to lower bacterial contamination?


Edited by MycoWill (04/29/20 11:13 PM)


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Re: Is this bacteria on this petri dish? [Re: MycoWill]
    #26637929 - 04/29/20 11:56 PM (3 years, 8 months ago)

Its probably from the syringe most likely. Try anti-bacterial agar. Or maybe experiment with spore solutions. Ive mixed open-air spores with water and a tiny amount of Lysol and produced clean PF Jars. It will probably work on Agar too.


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Offlinemushpunx
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Re: Is this bacteria on this petri dish? [Re: MycoWill]
    #26638143 - 04/30/20 02:49 AM (3 years, 8 months ago)

Quote:

MycoWill said:



I attempted to clone 5 different mushrooms from a MS tub recently and every one of my 15 petri dishes looks likes these. Are these all contaminated with a bacterial infection? And is it still possible to obtain a clean sample as the mycelium grows out?





Are these the initial plates with tissue from the clone or transfers?

When myc is sitting on a blob of bacteria that big I usually do not bother with a transfer.
It's possible you could transfer away clean myc, but those cultures, if it was me I wouldn't bother. 


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Edited by mushpunx (04/30/20 03:14 AM)


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Re: Is this bacteria on this petri dish? [Re: MycoWill] * 1
    #26638179 - 04/30/20 03:12 AM (3 years, 8 months ago)

Quote:

MycoWill said:
Thanks bks a ton for the quick advice, I will transfer it out a few times until I get a clean dish. Any tips on procedures to reduce this in the future? Would the bacteria have been more likely on the mushroom or introduced in the lab process? I left 3 dishes alone and they are still in front of my flow hood with no bacteria at all.

I have been have bacteria issues all around since day 1. However the plates that inoculated these grains were clean. Besides proper fae, is there anything else I can do to lower bacterial contamination?




If you are having issues with bacterial spawn, either your plates weren't as clean as you think or your sterile technique is off. (And sometimes it just happens to all of us).
You can have clean plates and still end up with bacterial spawn FYI. Bacteria or mold can get in when inoculating the grain or doing G2Gs if your technique isn't on point.

Bacteria in your agar work:

Bacteria or mold colonies often appear on germination plates, it's to be expected because spore prints (and especially syringes) are never 100% clean. Agar gives us a 2-D surface to see the contams and isolate away from them. If you keep seeing bacteria or mold in your plates after your first transfer or two, either your culture is contaminated or you have sloppy technique.

When you clone, are you tearing your fruits open by hand or slicing into them?
When you clone, you want to tear the fruit open by hand to expose the virgin tissue on the inside, and drag your flame sterilized scalpel to pick up a small piece of tissue to drop on your plate. If you slice in, since mushrooms are grown in open air and not sterile, you are pushing mold or bacteria from the outside of the mushroom into the inside.


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OfflineMycoWill
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Re: Is this bacteria on this petri dish? [Re: mushpunx]
    #26638528 - 04/30/20 08:22 AM (3 years, 8 months ago)

Quote:

mushpunx said:
Quote:

MycoWill said:
Thanks bks a ton for the quick advice, I will transfer it out a few times until I get a clean dish. Any tips on procedures to reduce this in the future? Would the bacteria have been more likely on the mushroom or introduced in the lab process? I left 3 dishes alone and they are still in front of my flow hood with no bacteria at all.

I have been have bacteria issues all around since day 1. However the plates that inoculated these grains were clean. Besides proper fae, is there anything else I can do to lower bacterial contamination?




If you are having issues with bacterial spawn, either your plates weren't as clean as you think or your sterile technique is off. (And sometimes it just happens to all of us).
You can have clean plates and still end up with bacterial spawn FYI. Bacteria or mold can get in when inoculating the grain or doing G2Gs if your technique isn't on point.

Bacteria in your agar work:

Bacteria or mold colonies often appear on germination plates, it's to be expected because spore prints (and especially syringes) are never 100% clean. Agar gives us a 2-D surface to see the contams and isolate away from them. If you keep seeing bacteria or mold in your plates after your first transfer or two, either your culture is contaminated or you have sloppy technique.

When you clone, are you tearing your fruits open by hand or slicing into them?
When you clone, you want to tear the fruit open by hand to expose the virgin tissue on the inside, and drag your flame sterilized scalpel to pick up a small piece of tissue to drop on your plate. If you slice in, since mushrooms are grown in open air and not sterile, you are pushing mold or bacteria from the outside of the mushroom into the inside.



Quote:

mushpunx said:
Quote:

MycoWill said:



I attempted to clone 5 different mushrooms from a MS tub recently and every one of my 15 petri dishes looks likes these. Are these all contaminated with a bacterial infection? And is it still possible to obtain a clean sample as the mycelium grows out?





Are these the initial plates with tissue from the clone or transfers?

When myc is sitting on a blob of bacteria that big I usually do not bother with a transfer.
It's possible you could transfer away clean myc, but those cultures, if it was me I wouldn't bother. 





They are, yes. I harvested the fruits, put them into a small totes I sanitized with alcohol and closed it. Took the tote to the lab and opened it in front of my flow hood. I tore apart a particular mushroom and cut out a chunk from the inside of the stem and placed it on the agar plate and then sealed with micro pore tape. Flame sterilizing between each mushroom. The fact that every single plate has bac on it tells me that my source must have had bac and that when I opened it in front of the hood it blew all over the room? Unless I'm making incorrect assumptions? I know the plate prep was fine because the empty plate I have in the same room are still good to go.


Edited by MycoWill (04/30/20 08:28 AM)


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Offlinemushpunx
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Re: Is this bacteria on this petri dish? [Re: MycoWill]
    #26638639 - 04/30/20 09:46 AM (3 years, 8 months ago)

"They are, yes. I harvested the fruits, put them into a small totes I sanitized with alcohol and closed it. Took the tote to the lab and opened it in front of my flow hood. I tore apart a particular mushroom and cut out a chunk from the inside of the stem and placed it on the agar plate and then sealed with micro pore tape. Flame sterilizing between each mushroom. The fact that every single plate has bac on it tells me that my source must have had bac and that when I opened it in front of the hood it blew all over the room? Unless I'm making incorrect assumptions? I know the plate prep was fine because the empty plate I have in the same room are still good to go."

Dude everything in open air has bacteria and mold spores on it, the mushroom you tore open most certainly does. That's why we never let anything that isn't sterile (gloved hands especially) come between the filter face and open sterile media.
As long as you don't let the mushroom come between the HEPA and your open pitre dish then any spores/bacteria on it won't blow into your plate.
In a still air box you don't move anything unsterile over top of open media, because it will settle down into it. In a flow hood the flow is blowing towards you in laminar flow, so you don't want possible contaminants to ever between the filter and your open media.

Your cloning procedure sounds right to me.
Don't know why you have bacteria but as you can see it's coming from the tissue you set on the plate, it's not all over the place.
Instead of cutting a chunk out, just tear the mushroom open and drag the end of the scalpel along the inside tissue and you should pick up a piece cleanly. Lift it up And into the sterile flow, making sure other parts of the mushroom don't come between the filter and the tissue on your scalpel.


  If I were you, I would abandon those clone plates, and start over. You'll get it.


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OfflineMycoWill
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Re: Is this bacteria on this petri dish? [Re: mushpunx]
    #26655931 - 05/07/20 07:56 PM (3 years, 8 months ago)



Update (I will provide updates and give my final thoughts as when I search old threads they tend to die out without a resolution which sucks for us new guys trying to learn)

As you can see there looks to be some decent growth going on. I took 3 samples and put them on 3 new plates last night and will update once they grow out or get infected and stall.


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Offlinemushpunx
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Re: Is this bacteria on this petri dish? [Re: MycoWill]
    #26655945 - 05/07/20 08:06 PM (3 years, 8 months ago)

I can't see the left plate so well but if you look at the right one, but do you see the bacteria peeking out from under the culture at 9oclock (under the writing) - also at 12oclock at top of plate and the edge of the plate from 2 to 5 o'clock.

Cultures don't always stall once infected


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OfflineMycoWill
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Re: Is this bacteria on this petri dish? [Re: mushpunx]
    #26655969 - 05/07/20 08:17 PM (3 years, 8 months ago)

There's definitely some bacteria left. I'm sure it'll take a few transfers to get a clean sample but there is hope I believe.


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OfflineKizzle
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Re: Is this bacteria on this petri dish? [Re: MycoWill] * 1
    #26656192 - 05/07/20 10:12 PM (3 years, 8 months ago)

You might want to consider making some lower nutrient agar or antibiotic agar to use when you're cloning. It makes it easier to isolate clean mycelium from any bacteria that came along with it and then you can transfer the clean mycelium to your normal agar.


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OfflineMycoWill
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Re: Is this bacteria on this petri dish? [Re: Kizzle] * 1
    #26656263 - 05/07/20 10:36 PM (3 years, 8 months ago)

Quote:

Kizzle said:
You might want to consider making some lower nutrient agar or antibiotic agar to use when you're cloning. It makes it easier to isolate clean mycelium from any bacteria that came along with it and then you can transfer the clean mycelium to your normal agar.




Thanks for the tip, I will absolutely try that next time.


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