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OfflineIramLex
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Need help / ressources about bacteria on the sides of petri dishes
    #27130665 - 01/06/21 02:28 PM (3 years, 1 month ago)

Hello there !

I am new to this hobbie, and I started agar 2 mounth ago.
I started with clones from fruit bodies that gave sectoring and I am trying to get a clean monoculture.

My problem is that I keep getting bacterial contamns on the edges of my plates. It's on nearly every plates...

I am on my fift transfer the myc is going well but I can't use theses plates until there is no bacteria.

I am using a still air box. I wipe everything with alcohol, Pressure cook my cleaned petri dishes (resusable PP5 dishes I am trying to reduce plastic wastes). And my agar is BRF agar, not the clearest agar but it seems to work.
I wrap my plates with plastic wrap before transfering something on it. I don't want to use too much parafilm (damn expensive).

Do you have ressources or advices to reduce this bacterial problem ?

These are my ideas :

- Find something that works better than alcohol.
- It might be because I have to open the stil air box to fit the plates in it (plates don't fit through arm holes).
- I put the plates directory on the "floor" if the SAB, do I need to level them up ?
- It could be my plastic wrap, as I usually cut slices of it outside of the SAB so they are not sterile. But parafilm is not sterile either and people seems to have no issues with it.
- I also do my transfers near the arm holes, not in the back if the SAB, I have shaky hands it is easier for me. But I have spotted some of these bacterial contamn before transfers.

Thanks a lot !


Edited by IramLex (01/06/21 02:36 PM)


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OfflineTigerStrike
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Re: Need help / ressources about bacteria on the sides of petri dishes [Re: IramLex]
    #27130709 - 01/06/21 02:44 PM (3 years, 1 month ago)

Does not matter if you need to lift your sab to put the sleeve in the sab.
Place inside EVERYHITNG YOU WILL NEED then wipe and follow proper sab tech.

You are getting bac landing on your plates from sitting open inside your SAB. You need to remove the sleeve place it somewhere perfectly clean and then slide the sleeve back over your plates when you are done. Air is wushing and whizzing into your plates from arm holes and drafts ect.

You can also place them in Quart gal ect zip lock bags like I do without any issues. The bags that come out of zip locks are produced very hot and they do not have many if any contaminated inside them.

according to rr in this thread "https://www.shroomery.org/forums/showflat.php/Number/6783584 Ziploc bags are pretty darn sterile thy are hot and fast and sealed.

My most important point is 1 DO NOT GIVE UP

anything else does not matter as long as you are not giving up!!!! You dont go!!!!! you stay and try again!!! I would like to see another pour at very low cooled off agar temps and then not letting plates sit inside your SAB more than hr or 2 max to remove condensation you will still loose some with this too will loose less if you cap and bag immediately but will have to deal with more condensation which is better than dealing with contaminated plates and does not cause any problems. until you pour about 3 or 4 more times as your aseptic tech gets better by just practicing


Edited by TigerStrike (01/06/21 03:04 PM)


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OfflinecronicrFacebook
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Re: Need help / ressources about bacteria on the sides of petri dishes [Re: IramLex]
    #27130715 - 01/06/21 02:47 PM (3 years, 1 month ago)

its likely something in your technique causing you problems, if its happening away from your transfer at every transfer at the edge of your no pours the only common denominator is where your scalpel is reaching over your work or what you do with your lid while making transfers


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OfflineIramLex
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Re: Need help / ressources about bacteria on the sides of petri dishes [Re: TigerStrike]
    #27130769 - 01/06/21 03:12 PM (3 years, 1 month ago)

Thanks for your support !

TigerStrike :
Okay so if I understand well, you are saying I should have covered my plates in the SAB just after pouring.
The problem is, these resusable PP5 petri dishes don't comes in sleeves, so I need to find another thing :/

Eventually I'll try ziplock bags but I feel bad about throwing out these bags after one use...

Be sure I wont give up, but I feel I lack something in my sterile techniques and I don't have found the solution that suits me.

cronicr:
What I do for now is to hold the top part of the dish with my left hand and do the transfer with the right hand. I think I sometimes touch the outside of the bottom part with my right hand, it is a problem ?


Edited by IramLex (01/06/21 03:21 PM)


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OfflineTigerStrike
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Re: Need help / ressources about bacteria on the sides of petri dishes [Re: IramLex]
    #27130799 - 01/06/21 03:26 PM (3 years, 1 month ago)

There are silicone Ziploc baggies and permanent versions of all the items you want to use.


With silicone bags, you can sterilize them in the PC if the temp withstands which it should.

There is a compromise between convenience of disposable vs washable and enviro conscious.

It warms my heart to see you so concerned with our world and I will be making a few simple changes to start stopping some of the plastic pollution I am committing also!!


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OfflineMarxcelium
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Re: Need help / ressources about bacteria on the sides of petri dishes [Re: TigerStrike]
    #27130836 - 01/06/21 03:45 PM (3 years, 1 month ago)

Are you getting lots of condensation on your plates when you pour that pools at the edges? Contams can can use the water as a pathway into the dish if you touch the edges when you're pouring or transferring. You'll end up with a ring of bacteria or whatever around the edge of the dish.


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OfflineIramLex
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Re: Need help / ressources about bacteria on the sides of petri dishes [Re: Marxcelium]
    #27130856 - 01/06/21 03:53 PM (3 years, 1 month ago)

Yes it is exactly what happens. Lots of condensation when pouring.
I get rings of bacteria on nearly every plates.
Maybe I should wait for agar to cool more ?


Edited by IramLex (01/06/21 03:54 PM)


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OfflineMarxcelium
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Re: Need help / ressources about bacteria on the sides of petri dishes [Re: IramLex]
    #27130958 - 01/06/21 04:37 PM (3 years, 1 month ago)

If you have a meat thermometer, place your hot, sealed bottle of sterilized agar in a tub of water that's at least 120f and then put the thermometer in the water.

Like this:



Most people say wait until the temp hits 120f and then pour, but I've found with my thermometer that I can wait until 112f for better results.

I then put hot water in blank plates that I keep around and put the blank plates on top of the stacks of poured plates to keep condensation from forming on the top agar plate.

You can also pour the plates, slide them to the back of your SAB, and cover you arm holes to let them dry for a couple days if you need to. 

You'll get rid of condensation this way and, hopefully, cut off the contams' pathway into your plates.

Really hope this solves it for you. Keep at it and you'll get there!


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OfflineTattersail
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Re: Need help / ressources about bacteria on the sides of petri dishes [Re: Marxcelium]
    #27131377 - 01/06/21 07:05 PM (3 years, 1 month ago)

I use pp5s as agar dishes too. I try to hold them upside down in the SAB whenever possible so that contams don't fall in. I haven't ever wrapped them with parafilm or anything and they're usually grand. Tons of condensation but it hasn't caused me bacterial issues so far.

Have you tried leaving a couple of the plates to see if they contam with no transfers?

Placing them on something above the floor of the SAB can help. I use the rack thingy from the PC. If the arm holes of the SAB arent big enough to get the plates through, perhaps they are creating more air movement than necessary abd bacteria is getting knocked off the edges?


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