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Mushrooms, Mycology and Psychedelics > Advanced Mycology
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Isolating cultures away from contaminates on agar
    #19205281 -

When it comes to cleaning up cultures on agar, is there a time when it's not even worth it to open the petri? 

In this image, I have an enoki culture that has what I believe to be a trich contamination.  My concern is that by opening this plate, even in my SAB, that I'm likely to just spread those spores and increase my chances of future trich contaminations in future work.



(There also looks to be bacterial contamination on the left side, possibly why the mycelium only grew to the right.  I'm not so worried about opening a plate with bacterial contamination, as it needs direct contact to spread, unlike mold which can sporulate far and wide...)

Is this something I shouldn't be worried about if, for example, I follow something like the following procedure?

- clean and "sterilize" SAB with an acidified bleach spray before working
- move ONLY the infected plate and the four or five additional plates I intend to transfer to into the SAB
- flame my scalpel and move in to SAB to do the first transfer form infected plate to new petri

then EITHER

- remove scalpel from SAB to re-flame and repeat above step

OR

- wipe or dunk scalpel blade with acidified bleach solution and then perform next transfer after 60 seconds of bleach contact?


The reason I mention the options for the repeating step is because I have noticed an increase in contamination when removing the scalpel to flame sterilize between doing transfers, compared to alcohol/bleach sterilization  between transfers.  My guess is that I'm bringing spores into the box when I take my arms out and put them back in.

Lastly, if i'm making several transfers from the same petri, to either isolate or expand it, is re-sterilizing the scalpel even necessary?

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Re: Isolating cultures away from contaminates on agar [Re: socraticd]
    #19205362 -

I wouldn't call this Advanced Mycology, and you'll get better results and answers in the Cultivation forum

:twocents:

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Re: Isolating cultures away from contaminates on agar [Re: OgreLokon]
    #19205470 -

So I'm gonna be a dick now, and observe that either you don't know the answer or even have a qualified opinion on the question.  OR you do know the answer or have a qualified opinion, but instead of being helpful you throw your proverbial "two cents" in to say that this isn't an "advanced" enough topic...  nice, and thank you.

If this isn't an "advanced" enough question for this forum, and you don't have any idea how to answer it, then WTF are you doing here?  Or, again, you DO have a qualified opinion or answer and are just choosing to turn your nose up entirely, rather than actually be helpful.

An answer ALONG WITH a quick opinion that I might have had more luck or more response in general cultivation or gourmet/medicinals would also have been worlds more helpful and/or less annoying.

Forgive me if I consider agar work, isolations, and slant work to be advanced compared to the bulk of the traffic on General Cultivation...  Either way, even if this is the "wrong" place for my post, your response provides nothing of ANY value to the forum as a whole.  Why bother?

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Re: Isolating cultures away from contaminates on agar [Re: socraticd]
    #19205585 -

No, I just think that what he meant was that you are likely to get a better answer to your question in the Mushroom Cultivation Forum.

Anyway, I see a Trichoderma contamination and a bacterial contamination in that plate, the Trichoderma has already sporulated which is evident because it has gone green, it is likely that if you attempt to do any transfers from that plate your new plates will be contaminated with  Trichoderma, if possible you should start a new culture, if you see any suspicious growth you need to make your transfers as soon as possible before you see any green.

As soon as you see growth from your spores or tissue transfer it to a new plate, you may need to do that a few times before it is clean.
Good luck:thumbup:


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Re: Isolating cultures away from contaminates on agar [Re: inski]
    #19205639 -

Yeah,  sorry I just have been working WAY too many hours lately and I'm a bit touchy.  I just never understand those types of comments I guess... :undecided:

Regarding the plate in question,  that was my suspicion as far as the right course of action.  I'm getting better at spotting the contams before they sporulate, so I'll try to stay ahead of them on future plates

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Re: Isolating cultures away from contaminates on agar [Re: socraticd]
    #19206040 -

You can do a series of fast transfers.  Use your glovebox, even if you have a laminar flow hood.  It really doesn't matter if you grab trich spores along with the enoki mycelium because you're going to transfer again in a few days before they have time to germinate. Transfer from the extreme leading edge of the mycelium growth every three days or so until you have a clean culture.
RR


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Download Let's Grow Mushrooms



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."
Thomas Edison

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Re: Isolating cultures away from contaminates on agar [Re: RogerRabbit]
    #19212248 -

I always assume that if there's visible mold in the dish that any transfers will have mold spores on them anyway. Then since you are expecting the mold you can know to transfer the new mycelium from the next culture before the mold has time to start growing.

No doubt some mold spores may become airborne in the box while opening the dish but you gotta do what you gotta do and I don't think opening a petri dish briefly in a SAB is going to have serious consequences to the outside air.


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Re: Isolating cultures away from contaminates on agar [Re: Kizzle]
    #19212653 -

As fas as I understand it yet, the mold spores would have to germinate on the new dish first, while the transferred mycelium would take off immediately. So the mycelium would outrun it before germination of the mold and be transferred to a new dish a few times leaving a clean mycelium culture.

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Re: Isolating cultures away from contaminates on agar [Re: monoculture]
    #19215183 -

Well I figure being able to transfer away from mold and clean up these types of cultures will be a good skill to have at some point in the future, so I'll give it a shot and report back.

Thanks guys!

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Re: Isolating cultures away from contaminates on agar [Re: socraticd]
    #19235433 -

Check this out
You know the crack pipes they sell in the ghetto, the ones with the rose in them.. I have a picture here..


Anyway remove anything from the glass and cap one end with polyfill held in with micropore tape, fill tube with brf or straw (I prefer straw), then clean and plug with polyfill, no tape on this end though


Sterilize then in a SAB remove the polyfill and place a wedge of agar from the culture you want to clean then replace the polyfill

The objective is to take advantage of trichs need to sporulate in order to spread efficiently, while taking advantage of cube mycelium long reach. Trich might bloom where you placed your wedge but what reaches the other side will be clean cube myc for you to work with.

This technique works great for most contams, trich being the example here

Also TEG (Triethylene glycol) will take care of those pesky trich spores that end up in your SAB


--------------------


AMU Q&A Thread because questions deserve answers.

Edited by WillSolvem (12/06/13 07:44 AM)

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Re: Isolating cultures away from contaminates on agar [Re: WillSolvem]
    #19235546 -

WillSolvem said:
Check this out
You know the crack pipes they sell in the ghetto, the ones with the rose in them.. I have a picture here..


Anyway remove anything from the glass and cap one end with polyfill held in with micropore tape, fill tube with brf or straw (I prefer straw), then clean and plug with polyfill, no tape on this end though


Sterilize then in a SAB remove the polyfill and place a wedge of agar from the culture you want to clean then replace the polyfill

The objective is to take advantage of trichs need to sporulate in order to spread efficiently, while taking advantage of cube mycelium long reach. Trich might bloom where you placed your wedge but what reaches the other side will be clean cube myc for you to work with.

This technique works great for most contams, trich being the example here

Also TEG (Triethylene glycol) will take care of those pesky trich spores that end up in your SAB


:sunspots:

Genius! Very nice method there, have to keep a note on that one.


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No, I wasn’t born in 98’


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Re: Isolating cultures away from contaminates on agar [Re: sharpshroomer98]
    #19235594 -

Hmm....

That's a pretty interesting idea.  I don't work with cubes, but I guess I don't see why this wouldn't work for edibles as well.  Especially the more aggressive strains.

Nice!  Crack-pipes here I come!!! :thumbup:

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