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OfflineBlake_Shroom
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Bacteria or "chunks" in petris
    #18911055 - 09/30/13 12:10 PM (10 years, 4 months ago)

Well i've poured a lot of petris before, but the last 2 times I poured them, they turned out like this




The time before, I poured the petris, let cool in front of flowhood, then immediate inoculated all that I poured. Then after making transfers, it seemed like there's bacteria in the petris (not on top) and it got transferred too.

Now I have all these petris that look like this, and I'm wondering what's up. I PC for 30 mins, usually more. I run my flowhood for a few hours before pouring, then pour, leave them uncovered, and place tops on them after they cool (i hate condensation on my petris tops).

I'm thinking that either I'm not properly PC'ing, or that some how bacteria fell from my body into my jar that I pour from, and that's how all my petris turn up like this.

Any input?


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OfflineOregonMushys
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Re: Bacteria or "chunks" in petris [Re: Blake_Shroom]
    #18911099 - 09/30/13 12:20 PM (10 years, 4 months ago)

Maybe clean the face of your flowhood and all working surfaces before turning on as well, and perform more proper sterile procedure? Maybe dont leave the dishes open while letting solidify. I can also see there is an invasive mold right at about 8'O'clock on your second pic.

If you set something very warm on top of the petri stack while solidifying, im pretty sure it will prevent condensation on the upper dishes during the cooldown stage. Though it will condense again if you get temp fluctuations during colonization.


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Edited by OregonMushys (09/30/13 12:26 PM)


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OfflineBlake_Shroom
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Re: Bacteria or "chunks" in petris [Re: OregonMushys]
    #18911153 - 09/30/13 12:39 PM (10 years, 4 months ago)

Yea, I know about that mold. It's wierd, growing under the agar on the bottom of the plate.

I'm thinking I'm not pc'ing long enough. I'm using a small pc which doesnt have a rocker, but I've used it so many times before and havent had issues.

I'm about to pc up some more agar, fully clean my work area with bleach water first then with alcohol, pc my agar in my aa so i know it'll be in there for the right amount of time, and pour my jars as neat as i can. Luckily I have 1 plate of my original MS plate so I can transfer from that rather than from these tainted plates.

I'm also going to pc my tyvek sleeves in some jars and open it infront of the flowhood so its sterile. I have plenty of space in my pc so i might as well lol


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OfflineRogerRabbitM
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Re: Bacteria or [Re: Blake_Shroom]
    #18911194 - 09/30/13 12:50 PM (10 years, 4 months ago)

Quote:

Blake_Shroom said:


Now I have all these petris that look like this, and I'm wondering what's up. I PC for 30 mins, usually more. I run my flowhood for a few hours before pouring, then pour, leave them uncovered, and place tops on them after they cool (i hate condensation on my petris tops).





This is your problem; bad technique.

Always put the lids on the petri dishes immediately after pouring.  Never have a dish open for more than 5 seconds.  There will be no condensation by the next day if you stack all the dishes into a single stack after pouring as shown in the agar-petri dish video. 

No flow hood is 100% perfect.  With up to a few hundred cubic feet per minute, each cubic foot of air having tens of thousands of contaminant particles/spores @ 99.99% efficiency, think of how many contaminants are being blown through the hood and into your dishes as they sit open.

Quote:

Blake_Shroom said:
Yea, I know about that mold. It's wierd, growing under the agar on the bottom of the plate.




This is from exposure with the lids off prior to pouring, allowing contaminants to settle on the empty petri dish.

That being said, most of what I see above appears to simply be undissolved solids from your nutrients.
RR


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OfflineBlake_Shroom
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Re: Bacteria or "chunks" in petris [Re: RogerRabbit]
    #18911214 - 09/30/13 12:57 PM (10 years, 4 months ago)

Okay, I'll put the lids on as soon as i pour this time.

And how do i make sure all of these nutrients dissolve fully? Am i'm putting in too much Malt extract?

I always use warm water and stir my MEA solution before PC'ing so that the MEA is mixed up evenly throughout the entire solution. I do roughly 5 grams ME to 5 grams Agar to 1 pint of water, which pours 40 petris for me (60mm).

I looked up agar recipes long ago and have been using this mixture for a while with no issues


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OfflineRogerRabbitM
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Re: Bacteria or "chunks" in petris [Re: Blake_Shroom]
    #18911235 - 09/30/13 01:03 PM (10 years, 4 months ago)

If you're using agar-agar from the grocery store you should boil all the ingredients together to ensure they're mixed, then load into your bottle and sterilize.
RR


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OfflineBlake_Shroom
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Re: Bacteria or "chunks" in petris [Re: RogerRabbit]
    #18911351 - 09/30/13 01:36 PM (10 years, 4 months ago)

http://www.amazon.com/Molecule-R-Agar-1-Pound/dp/B005IGBQ0Y/ref=sr_1_4?ie=UTF8&qid=1380569752&sr=8-4&keywords=agar

That is what I use. Next batch of petris i'll boil first to make sure it's properly mixed.

Thanks for the help


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Invisiblebarong
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Re: Bacteria or "chunks" in petris [Re: Blake_Shroom]
    #18911456 - 09/30/13 02:07 PM (10 years, 4 months ago)

Quote:

Blake_Shroom said:

I always use warm water and stir my MEA solution before PC'ing




TIP: Malt Extract dissolves better in cold water. Tr that, the microwave for a few 20 second bursts to dissolve your agar,stirring between cycles. Watch out for overboil though. Youll get the hang of it after the first spill.


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