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Offlinehallmaker
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Re: i keep getting bacteria in my agar dishes [Re: InfiniteDreams]
    #26548926 - 03/21/20 01:21 PM (3 years, 10 months ago)

What is ISO?


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OfflineBooShow
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Re: i keep getting bacteria in my agar dishes [Re: hallmaker]
    #26548929 - 03/21/20 01:23 PM (3 years, 10 months ago)

Quote:

hallmaker said:
What is ISO?



Isopropyl Alcohol


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OfflineLittleBoard
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
    #26549082 - 03/21/20 03:02 PM (3 years, 10 months ago)

Quote:

Mycobum7 said:
i said this once but ill say again, last time i didnt warm the water up much and got a lot of sediment, so i poured off what was dissolved and threw out some sediment, maybe i should just leave it next time?



At which step did you do this and why? I assume before PCing. I imagine that this can throw of your ratios and you get "watery" agar.
What is your friend's procedure where there is no contam with apparently all factors the same as yours?


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OfflineSockadin
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Re: i keep getting bacteria in my agar dishes [Re: LittleBoard]
    #26549188 - 03/21/20 03:53 PM (3 years, 10 months ago)

So he is ordering pre poured plates and getting bacteria? I didn't realize this. I have no experience in the matter, but if you let someone else make your plates why not let them grow your shrooms?


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OfflineKizzle
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
    #26549281 - 03/21/20 04:54 PM (3 years, 10 months ago)

Quote:

Mycobum7 said:
statements like yours really make me question my dishes



That's the first thing I'd suspect, all things considered. Pouring is relatively easy so if you're not having problems when doing transfers I would be surprised if it was your sterile technique or flowhood that's the problem. You could do a test and try pouring some into a sterile mason jar or sterile PP container and see if it still happens with that. I've never heard of anyone getting presterilized dishes that were compromised so I don't know how likely it is.

Something else to consider though is if the plates your friend made were made with sabouraud agar or antibiotic agar it might prevented the bacteria from showing up.


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InvisibleInfiniteDreams
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Re: i keep getting bacteria in my agar dishes [Re: LadysKnight]
    #26549335 - 03/21/20 05:27 PM (3 years, 10 months ago)

Quote:

LadysKnight said:
Quote:

InfiniteDreams said:
I use pour plates. 
They come sterile in a sleeve. 
I sterilize my agar in the PC before I pour. 
I pour in a SAB. 
I do not use ISO. 
I do not use gloves. 
I do wash my hands.
I do not wrap the plates.
After they have set, I do put them in ziplock bags to store on a shelf until time for use.

I have not yet found a single case of contamination when I go to use them, sometimes weeks later.




Hey InfiniteDreams, I'm also on team Ziploc, no wrap, and store in the open.
When using the plate, I wipe the baggie with iso outside the sab, open baggie in sab, toss baggie.
So you don't wipe the baggie at all?
Do you open it inside or outside sab?




At first I did that also.  Now I don't use iso.  I also am reusing the same ziplocks.  The ones I"m using have a slide close and can fit about 20 plates in a single bag.  I've been opening the bag outside the SAB. But I am right in front of it and pass the stack of plates through the hole and set them on the rack inside my SAB. The plates themselves remain closed the entire time.  After they are inoculated I will wrap them with parafilm.


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InvisibleLadysKnight
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Re: i keep getting bacteria in my agar dishes [Re: InfiniteDreams]
    #26549387 - 03/21/20 05:54 PM (3 years, 10 months ago)

Ok, I see, not too similar, mine go from PC to sab into individual ziplocks after inoc. I may still try taking the plate out before sab.


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OfflineMycobum7
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Re: i keep getting bacteria in my agar dishes [Re: Mycobolical]
    #26549500 - 03/21/20 06:45 PM (3 years, 10 months ago)

this is great information, thank you. i am wrapping my dishes with parafilm, i use two squares, fold  once hot dog way so double layered, and wrap. nicely so it seals the overlap with no tears, this is done in front of the hood.


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OfflineMycobum7
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
    #26549504 - 03/21/20 06:48 PM (3 years, 10 months ago)

no these are not pre poured plates, i am pouring. i think i will do another pour tonight with the best tek i have yet done, keeping in mind things like moisture content / condensation and see what happens.


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OfflineMycobum7
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Re: i keep getting bacteria in my agar dishes [Re: PiptheGreAtest]
    #26549514 - 03/21/20 06:55 PM (3 years, 10 months ago)

can you drop a link to the c100


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OfflineSockadin
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
    #26549561 - 03/21/20 07:22 PM (3 years, 10 months ago)

What is your recipe? Are you venting the PC for long enough? I'm grasping at straws here man you got a good puzzle on this one.

Thought I just had. Make a batch of agar and don't pour it. PC it and let it cool in the bottle till it gets all solid infront of the FH. Wait a few days. If you get bacteria in the bottle you can start there and work backwards. If it doesn't just rePC and work forwards


Edited by Sockadin (03/21/20 07:24 PM)


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OfflineMycobum7
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Re: i keep getting bacteria in my agar dishes [Re: Sockadin]
    #26549859 - 03/21/20 09:55 PM (3 years, 10 months ago)

i was thinking something like that, i was thinking maybe doing a sterilized jar and pouring into that to control jar vs. dishes.

When you say venting the pc what exactly do you mean and what is its importance?

recipe is 10 g agar 10 g malt per 500 ml, seems to be a relatively standard recipe. doing a cook right now and gonna do some cleaning and do a pour,

im thinking ill try to leave a small amount of agar in the container and see if any bacteria grown in there, the idea being having a control between a PC container and a sterile dish, although its not perfect its a place to start.


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OfflineMycobum7
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
    #26549893 - 03/21/20 10:16 PM (3 years, 10 months ago)

been reading lots on altitude, i live at about 5 k and have been doing standard pc times

anyone with info on this?

should i mod the pc or change times to account for this?


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OfflineSockadin
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
    #26549908 - 03/21/20 10:26 PM (3 years, 10 months ago)

Don't mod the PC.

When you are up to temperature you want to leave the weight off for 5 or so minutes until it stops spitting bursts or air and gives a full set of steam blowing out of the weight vent for 5 minuets. Displaced air can make your PC run worthless.

Also would be good to check on the flowhood pressure via a lighter. You want to see what the bend is because to much and you create turbulant air, to little and you don't create laminar flow.


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OfflineKizzle
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7] * 1
    #26549922 - 03/21/20 10:38 PM (3 years, 10 months ago)

Just run though the possibilities and check for whatever you haven't ruled out yet.

1. The agar is not being completely sterilized
2. The petri dishes are not sterile
3. Your hand is dropping contaminants onto the plate when you pour
4. The outside of the bottle is dropping contaminants onto the plate when you pour
5. The inside of the agar bottle is being contaminated as you open it
6. Your hand is touching the inside of lid while opening the petri dish
7. The flowhood is producing turbulence instead of laminar flow
8. A contaminated surface is coming between the front of the flowhood and the petri dish while the lid is open
9. A contaminated surface is coming between the front of the flowhood and the bottle while the bottle is open
10. Mites are getting into the culture

Like InfiniteDreams said, I can't recall a single time a plate contaminated before it was inoculated with exception of the period when I had a mite problem. If that's the case you'll see little trails on the agar like this.


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OfflineSockadin
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Re: i keep getting bacteria in my agar dishes [Re: Kizzle]
    #26549932 - 03/21/20 10:44 PM (3 years, 10 months ago)

Wow that is amazing. As always if you got contams ask kizzle.

Also how soon are you opening your PC after it cools? You can open to soon and cause issues there.


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OfflineMycobum7
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Re: i keep getting bacteria in my agar dishes [Re: Sockadin]
    #26550100 - 03/22/20 02:09 AM (3 years, 10 months ago)

ok i poured 20 new dishes and i feel this is my best pour session yet. what a cluster fuck this has been.

the first time the mix was likely off and was super hot when i poured it which just sucked. lots of excess moisture

second time, i let it cool too long and it was solidifying as i did the plates, was messy, tried to re heat it, container is too tall for microwave....i digress..

THHIRd time.. they say third is a charm .. i fucking hope. i poured 20 plates with what i consider to be ideal technique, nice and even pours and less moisture.


i will update in 72 hours either stoked about no bacteria or super frustrated and confused about the bacteria...


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Invisibletramalot
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Re: i keep getting bacteria in my agar dishes [Re: 99.99]
    #26550255 - 03/22/20 06:03 AM (3 years, 10 months ago)

Quote:

99.99 said:
Quote:






Thear is nothing wrong with my process now ,works fantastic but for your information I sterilize the agar in my pressure cooker for half an hour inside a beaker then I pour into the agar dishes while its still hot
let cool inoculate then wrap



Shouldn't you use a media bottle with a cap? An open beaker is not a common method!


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OfflineSockadin
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Re: i keep getting bacteria in my agar dishes [Re: tramalot]
    #26550420 - 03/22/20 08:30 AM (3 years, 10 months ago)

I use to use old whisky or wine bottles with an aluminum foil homemade lid with no issues. As long as you are keeping the lid on until in the SAB or FH it will be fine. Same thing for the breaker, if you are using a cover of some kind it should be fine.


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OfflineMycobum7
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Re: i keep getting bacteria in my agar dishes [Re: Sockadin]
    #26556630 - 03/25/20 09:00 AM (3 years, 10 months ago)

update....this pour appears to have no bacterial growth yet. its been about 3 days or so. i think this is what i learned..

too much moisture may possbly wick bacteria into the dishes

in the end i think the bacteria may have actually come from old parafilm

i had been given some parafilm from a friend and it didnt seem to adhere and peel off the backing as nicely as new parafilm, and after considering all things i believe the excess moisture combined with shit parafilm was an issue...


just saw that quite a few people looked at this post so thought i would share what i learned from it.


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