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How to deal with what I assume is bacterial agar contam.


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#1 DetritusTheEgo

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Posted 10 October 2020 - 08:31 AM

:cool: hello there,

 

I'm cleaning up some PS Mexicana Chicon Nindo on MAE agar. The source of my spores was from a swab so I knew that I'd have some cleanup work on my hands.

 

I did the swab to four plates. Two of the four swab plates had less contams then the others so I transferred those two plates to four plates each which was transfer #1. From those eight plates one was less contaminated the the others so I transferred that plate to eight plates for transfer #2. I'm currently still on transfer #2 and average contam rate is the same as transfer #1. I've got one plate that is less contaminated than the others. I still have the majority of all of these plates on hand.

 

In between transfer #1 and transfer #2 I made my batch of eight MAE plates and incubated them for seven to ten days at 70-80F to ensure that there was no growth and I had confidence in my jars and sterilization times. After that period nothing was growing on any of the plates so I proceeded.

 

I keep having a contamination that starts the edge of my jars then works it's way towards the center of the plate where the agar wedge transfer is. I assume that this is bacteria but I'm no contam expert. Any ideas?

 

1.jpg 2.jpg

 

Any guidance or input on how you would proceed? I don't mind doing transfer after transfer at this point but figured to reach out.

 

I can obtain some antibacterial agar. I'm also thinking about pressure cooking some of the MAE agar I already have in a beaker and pouring a thin layer over the plates that I've done transfers from so far, two from swab, one from T#1, and one from T#2. I've heard that the mycelium will pop up through the thin agar layer after a few days and the heat of the agar and suffocation slows the bacteria down during that time. Then do a transfer from the mycelium that pops through hopefully not taking any of the thin layer of agar with.


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#2 Boebs

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Posted 10 October 2020 - 12:22 PM

If its starting at the edge every time..
Then your plate is not clean.
Looks like your using Tupperware.
Plastic is very porous and can hold alot of contaminants.


If it was from your swab it would show contamination at the same location you swipe..
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#3 DetritusTheEgo

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Posted 11 October 2020 - 05:26 AM

If its starting at the edge every time..
Then your plate is not clean.
Looks like your using Tupperware.
Plastic is very porous and can hold alot of contaminants.


If it was from your swab it would show contamination at the same location you swipe..

I thought the same thing as well based on the location of the contam in relation to the wedge transfer. I am using PP5 lids and containers, I'm doing these in the no-pour fashion, and using SAB for my clean work.

 

I verified that my plates I used were clean though by incubating them at 70-80F for seven+ days before doing transfer #1 to transfer #2. After seven+ days none of the plates had any growth on them so I went through with the transfers and in a few days the mycelium as well as the contamination started showing.



#4 coorsmikey

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Posted 11 October 2020 - 09:39 AM

So then they are contaminating when you are opening the lid to inoculate. Are they under vacuum when you go to inoculate? If so try and mitigate the sudden change of pressure when opening. perhaps wrap the container with an alcohol soaked paper towel to work like a filter when sucking in the air. Things like scrubbing and sanitizing under your fingernails may even help. If you are working in a SAB then consider practicing your movements until they become smooth and fluid like. For me anyway, I have better luck in open air as I can move less clumsily  with smooth quick methodical movements without disturbing the surrounding air very much. I can't do that in a SAB or glovebox. If none of that jives, then I would consider using paradigm around the lids. if there is no vacuum when you open your no-pours then the lids aren't sealing and fluctuation in temps will force air in and out. Parafilm will help prevent the nasties from getting in if that's the case.


Edited by coorsmikey, 11 October 2020 - 09:58 AM.

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#5 Ferather

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Posted 11 October 2020 - 09:43 AM

Looks like the container wasn't sterilized, else you should see spots on the whole face of the agar, I only see growth coming from the plastic.


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#6 DetritusTheEgo

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Posted 12 October 2020 - 08:11 AM

So then they are contaminating when you are opening the lid to inoculate. Are they under vacuum when you go to inoculate? If so try and mitigate the sudden change of pressure when opening. perhaps wrap the container with an alcohol soaked paper towel to work like a filter when sucking in the air. Things like scrubbing and sanitizing under your fingernails may even help. If you are working in a SAB then consider practicing your movements until they become smooth and fluid like. For me anyway, I have better luck in open air as I can move less clumsily  with smooth quick methodical movements without disturbing the surrounding air very much. I can't do that in a SAB or glovebox. If none of that jives, then I would consider using paradigm around the lids. if there is no vacuum when you open your no-pours then the lids aren't sealing and fluctuation in temps will force air in and out. Parafilm will help prevent the nasties from getting in if that's the case.

 

Thanks for the advice coorsmikey.

 

I have had these specific containers pull a vacuum and suck the walls in if I remove them from the pressure cooker as soon as the stopcock falls and tighten down the lids. I thought about the fact that it would suck air into the container whenever I opened it and have since done my no pour agar plates in the evenings and then let them cool overnight inside the unopened pressure cooker. In the morning everything is room temp and I go through and tighten the lids as I remove them which averts the vacuum.

 

I will start using parafilm or cling wrap around where the lids meet the containers as I've not been doing that. I do have micropore tape as well. I will practice and run through my sterile method observing for anything that can be improved. That's a good practice even when not having issues.






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