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OfflineMoopers
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Registered: 05/08/20
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Sterile agar inoculation - unwrapped plates?
    #26679243 - 05/18/20 04:02 PM (11 days, 13 hours ago)

Hi all! Thanks for your responses on my last post.

I purchased some agar plates from an online vendor, and they arrived in a ziploc bag, no parafilm or other wrapping. I have a question or two about keeping things sterile/avoiding contam that I couldn't find an answer to in any of the agar teks, which assume you pour your own.

I have an inoculation loop and spore syringe, torch to sterilize. The actual plating of the solution and scratch plate creation I'm comfortable with.

Should I:

  • put the ziploc bag in my SAB before spraying lysol, then open inside? Concern: any contam in the bag enters the SAB.
  • open the bag outside of the SAB, and carefully place the plates inside, then close the SAB and spray disinfect? Concern: plate lids are loose, could pick up contam outside the SAB
  • close and spray the SAB, then bring the plates in through the arm holes? Same concern as above.
  • do another method you use as a best practice


Once the zip lock bag is open, can I keep storing plates in it in the fridge, or do I need to store them some other way?

Many thanks!


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OfflineCosmicKramer
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Registered: 10/23/12
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Re: Sterile agar inoculation - unwrapped plates? [Re: Moopers]
    #26679432 - 05/18/20 05:29 PM (11 days, 12 hours ago)

Only the inside of the plates need to be sterile, so you don’t have to be crazy with your tek.  I get my SAB ready, I only wipe it with water.  Wash my hands well and then open my bag and remove the plates I need and place them in the SAB.  Be purposeful so the lids don’t slide.  Remove the lid for the quick second it takes to noc.  With a spore syringe, I place one drop at the top of the plate and then use my loop to scrape it in a zig zag pattern down the remainder of the plate. Replace lid and slide it away.  Avoid passing anything over your plate other than your syringe needle and loop.  Finish remaining plates.  Then, wrap them with paraffin or just a roll of plastic wrap that has been cut to about an inch.  I sometimes fling my hands while trying to break the wrap so I do them last.

When you buy them sterile, they do not come with any seal on individual plates, just the bag Is sealed.  Good luck!


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OfflineMoopers
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Registered: 05/08/20
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Re: Sterile agar inoculation - unwrapped plates? [Re: CosmicKramer]
    #26679743 - 05/18/20 08:04 PM (11 days, 9 hours ago)

Thanks for the help and reassurance, Cosmic & LikeMyc! I tend to overthink things, and I've seen so many ugly plates on here and reddit, I hope I will do better.


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OfflineTrippyMomp
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Registered: 04/16/20
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Re: Sterile agar inoculation - unwrapped plates? [Re: CosmicKramer]
    #26679843 - 05/18/20 08:57 PM (11 days, 8 hours ago)

Quote:

CosmicKramer said:
Only the inside of the plates need to be sterile, so you don’t have to be crazy with your tek.  I get my SAB ready, I only wipe it with water.  Wash my hands well and then open my bag and remove the plates I need and place them in the SAB.  Be purposeful so the lids don’t slide.  Remove the lid for the quick second it takes to noc.  With a spore syringe, I place one drop at the top of the plate and then use my loop to scrape it in a zig zag pattern down the remainder of the plate. Replace lid and slide it away.  Avoid passing anything over your plate other than your syringe needle and loop.  Finish remaining plates.  Then, wrap them with paraffin or just a roll of plastic wrap that has been cut to about an inch.  I sometimes fling my hands while trying to break the wrap so I do them last.

When you buy them sterile, they do not come with any seal on individual plates, just the bag Is sealed.  Good luck!






I agree with the above. I wouldnt overcomplicate it. It's better for me if I take them out of the bag first. Maybe that's bad and not sterile but I feel like I'm more apt to hit the sides of my sab and mess up. Having just a few things in your sab is very helpful theres not a ton of room generally.


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OfflineSpaghettio73
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Registered: 03/13/20
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Re: Sterile agar inoculation - unwrapped plates? [Re: TrippyMomp]
    #26689364 - 05/22/20 11:05 PM (7 days, 6 hours ago)

I take it these were pre-poured? Are you certain they are sterile?

I prep my SAB with the lid as the base. Lined with paper towel I dowse with bleach water. After wiping down the inside of the tub with iso then spraying with water I let it sit on its lid as if I were ready to use it.  Shut off the a/c and fans. Wash my hands and spray with iso. From there I wipe the sleeve containing the dishes cut the sleeve at the bottom then turn the tub on its side. Holding one hand over the cut open sleeve I dispense plates in piles on the bleached paper towels. If I’m not using them all I reseal the sleeve by twisting the empty portion up like a bread bag. Tilt the tub back in place. Then heat seal or tape up the remaining dishes in the sleeve.

When you are working in the box run iso paper towel around the edge of the pitri making sure to get the sides of top, bottom and the lip. I don’t worry about setting anything down on the paper towel because they are wet with diluted bleach. Now I can work with the lid and nothing slides around.

Reverse order of this process when I parafilm the dishes in open air on the paper toweled lid.


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OfflineMoopers
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Re: Sterile agar inoculation - unwrapped plates? [Re: Spaghettio73]
    #26690136 - 05/23/20 10:57 AM (6 days, 18 hours ago)

Quote:

Then heat seal or tape up the remaining dishes in the sleeve.



Thanks for the tip. I'll do this when my next set of plates arrive. They have gentamicin in them, so hopefully that will give me a little buffer.

I went ahead an inoculated 4 of the first set of plates, but didn't seal the others after (didn't think of it). The other 6 I kept in their bag and returned them to the fridge. Two of those 6 plates show some spots that are likely bacterial contamination. I assume they're not able to be saved? I went ahead an inoculated them anyway with some Ps. galindoi from a MS syringe hoping I can transfer to clean when the new plates arrive, but I'm not hopeful. The other 4 I inoculated using spores from a print of Ps. cyanescens.

Wish me luck!


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