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A handy method to remove bacterial contamination from fungal cultures * 1
    #29657517 -

The article "A handy method to remove bacterial contamination from fungal cultures" by Shi, et al, 2019, is something that people on the shroomery might benefit from.

Quote:
Contamination control and removal are very important technical aspects of microbiological research. Bacterial contamination is very common in fungal cultures. Currently, the commonly used approach for inhibiting bacteria is antibiotic treatment; however, there are drawbacks to using antibiotics, including incomplete removal, limited antibacterial spectra, tendency toward recontamination, effects to fungal strains, and potential risks to the environment. Therefore, in the present work, we developed a new method for bacterial removal from fungi cultured on solid medium, the Cabin-Sequestering (CS) method, based on the different culture characteristics between fungi and bacteria. First, 3–5 mm round or square holes (the “cabin”) are excavated on a solid medium plate. The fungal strain containing possible bacterial contamination is inoculated into the cabin. The cabin is then covered with a sterilized coverslip, followed by incubation at the appropriate temperature. After 7–10 days of culturing, fungal hyphae grow out along the edge of the coverslip; however, the contaminating bacteria cannot pass through the space formed between the medium and the coverslip and, thus, remain in the cabin. The newly grown fungal hyphae around the coverslip are re-inoculated into fresh culture plates, where they form bacteria-free fungal colonies. The CS method is easy handling, with a short experimental cycle and rare recontamination. When necessary, it can also be used in combination with antibiotics in bacterial removal operations.





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Re: A handy method to remove bacterial contamination from fungal cultures [Re: CreonAntigone]
    #29657518 -

Cabin sequesters work great.

Several people here make their first or second transfer after spore into a CS.

It will get rid of all but the most stubborn bacteria. It can be a bit intimidating to new users, but is 100% worth the effort to learn.


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I've got skills, they don't know where to use me
I'm like the best dressed guy at the nude beach
Nice to meet you!

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Re: A handy method to remove bacterial contamination from fungal cultures [Re: PuffM4gicDr4gon]
    #29657522 -

So a coverslip is basically a glass slide right


--------------------
Why try for high fives,
When my pies have nine lives?

CAKE WARS III

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Re: A handy method to remove bacterial contamination from fungal cultures [Re: langten69] * 1
    #29657525 -

Some people use that. I just cut a chunk of agar and put it on top. Sometimes I'll even take a chunk from the same plate as long as I'm confident that I didn't bring any mold along with me


--------------------
I've got skills, they don't know where to use me
I'm like the best dressed guy at the nude beach
Nice to meet you!

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Re: A handy method to remove bacterial contamination from fungal cultures [Re: PuffM4gicDr4gon]
    #29657527 -

:coolpost:


--------------------
Why try for high fives,
When my pies have nine lives?

CAKE WARS III

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Re: A handy method to remove bacterial contamination from fungal cultures [Re: langten69]
    #29657610 -

Coverslips are placed on top of microscope slides. They are much thinner than the slides.

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Re: A handy method to remove bacterial contamination from fungal cultures [Re: The Tao]
    #29657611 -

nice creon, ty for this.f :rockon:


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guides | grows | supreme air box | sporeaddict

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Re: A handy method to remove bacterial contamination from fungal cultures [Re: CreonAntigone]
    #29657736 -

That's a neat idea.  I've heard of a technique where the established fungal culture on agar is covered with a new agar layer, which should slow the bacteria down while the fungal mycellium grows through the new agar.  The technique described here seems to work on the same principle.

A cover slip could certainly be flame sterilized without a problem.

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Re: A handy method to remove bacterial contamination from fungal cultures [Re: John in WI]
    #29657738 -

I remember this as the agar sandwich method. Works pretty good, used it a few times over the years.

https://www.shroomery.org/forums/showflat.php/Number/2894662

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Re: A handy method to remove bacterial contamination from fungal cultures [Re: Pastywhyte]
    #29657757 -

Cabins are great for getting rid of bacteria.



We already have a good thread about it though:
A trip to the cabin


--------------------
“All Fungi are edible.

Some fungi are only edible once.”
― Terry Pratchett


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Re: A handy method to remove bacterial contamination from fungal cultures [Re: John in WI]
    #29657764 -

I wouldn’t flame sterilize a glass coverslip. They’re pretty fragile.

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Re: A handy method to remove bacterial contamination from fungal cultures [Re: The Tao]
    #29657798 -

I missed the coverslip angle. That’s so stupid. The traditional sandwich method used a pane of agar cut from a different plate. It works well.

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Re: A handy method to remove bacterial contamination from fungal cultures [Re: Kloakk]
    #29657928 -

Kloakk said:
Cabins are great for getting rid of bacteria.



We already have a good thread about it though:
A trip to the cabin



Just trying to mentally process this image, it looks like you put a contaminated wedge in the middle of a cut out section of agar (cabin?) Then put a glass slide (coverslip?) On top of it and only the mycelium crawls its way onto the clean agar, correct me if im wrong


--------------------
Why try for high fives,
When my pies have nine lives?

CAKE WARS III

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Re: A handy method to remove bacterial contamination from fungal cultures [Re: langten69]
    #29657945 -

You take a potentially contaminated wedge and the cabin gives the mycelium room to outrun bacterial contamination then you transfer from that.
Click on the link Kloakk provided to follow the discussion.

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Re: A handy method to remove bacterial contamination from fungal cultures [Re: The Tao]
    #29657967 -

Ive been digging into that thread kloakk linked, on it he posted two pics

Quote:





Just curious what that golden ring on the second pic is cuz its not in the first


--------------------
Why try for high fives,
When my pies have nine lives?

CAKE WARS III

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Re: A handy method to remove bacterial contamination from fungal cultures [Re: langten69]
    #29658083 -

langten69 said:
Kloakk said:
Cabins are great for getting rid of bacteria.



We already have a good thread about it though:
A trip to the cabin



Just trying to mentally process this image, it looks like you put a contaminated wedge in the middle of a cut out section of agar (cabin?) Then put a glass slide (coverslip?) On top of it and only the mycelium crawls its way onto the clean agar, correct me if im wrong



You're not wrong. It's a glass slide I cut in half.

langten69 said:
Ive been digging into that thread kloakk linked, on it he posted two pics

Quote:





Just curious what that golden ring on the second pic is cuz its not in the first



The "ring" is water.


--------------------
“All Fungi are edible.

Some fungi are only edible once.”
― Terry Pratchett


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Re: A handy method to remove bacterial contamination from fungal cultures [Re: Kloakk]
    #29658094 -

Ah ok, im assuming that its a natural byproduct of the whole process then


--------------------
Why try for high fives,
When my pies have nine lives?

CAKE WARS III

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Re: A handy method to remove bacterial contamination from fungal cultures [Re: langten69]
    #29658130 -

langten69 said:
Ah ok, im assuming that its a natural byproduct of the whole process then



Yeah, mycelium produce heat so you're bound to get some water, especially with the glass roof.


--------------------
“All Fungi are edible.

Some fungi are only edible once.”
― Terry Pratchett


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Re: A handy method to remove bacterial contamination from fungal cultures [Re: Kloakk]
    #29658163 -

If the glass slides are too vulnerable to flame sterilize, how would you sterilize them? Could they be put in foil and put through a pc cycle?

The agar sandwich method seems to be an easier implementation of this.

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Re: A handy method to remove bacterial contamination from fungal cultures [Re: CreonAntigone]
    #29658165 -

CreonAntigone said:
If the glass slides are too vulnerable to flame sterilize, how would you sterilize them? Could they be put in foil and put through a pc cycle?

The agar sandwich method seems to be an easier implementation of this.



I haven't tried flame sterilizing the slides, I wrap them in foil inside a PP5 container and run them in the pressure canner when I do agar.



The Tao was talking about coverslips, they are smaller and a lot thinner. Nothing wrong with using agar roof as well, I personally just think it's easier to use slides.





--------------------
“All Fungi are edible.

Some fungi are only edible once.”
― Terry Pratchett


Edited by Kloakk (08/31/26 02:22 AM)

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