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OfflineBigSquid
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Help with Contam Identification
    #28178123 - 02/08/23 06:29 PM (1 year, 11 months ago)

Hello! My entire first attempt at agar was very quickly lost to this contamination-- I checked over the sticky thread on contam-I.D., but was having trouble placing it. I think it might be a type of yeast?





((These plates got a drop of MSS on Jan 11th, the contam popped up within 3-4 days, photo taken on Jan 15th))


I've run several more batches and transfers since ((with heavily improved sterile technique)) , and have only had a few problem cups so far. Noticeably, they've mostly been with the same syringes, and, spore swab I got from the same place. However, I recently had a few cups I used a LC on that has given me very good results so far crop up with a similar issue.



((One of the recent cup contams from 01/29-- a big thing I keep seeing is the, like, submerged and worm-like/tunnel appearance thats very visible from the sides))


Could I get some help ID'ing this? -- I'd like to check and see if I got a solid identification on it If I could pinpoint where in my process I need to pay extra attention to to keep sanitary. Any help is appreciated!

Its not a persistent problem through every cup in a batch I make, so I don't think it's the agar itself.

I keep a few control cups with every batch, now. Two that gets exposed to the same conditions as the others -- just without the mss/lc/print. And two or so cups that I keep fully unopened to see if anything pops up on the agar without any fiddling. This shit coming up on my first batch scared the hell out of me.

Thanks!

Edited by BigSquid (02/08/23 06:40 PM)

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OfflineMonkeyJesusFresco
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Re: Help with Contam Identification [Re: BigSquid]
    #28178496 - 02/08/23 10:37 PM (1 year, 11 months ago)

looks like my pet slime mold that recently ran away...

tell ya what,
is there anyway you can take them out of the bag and get photos?
(don't open the containers, of course)

the first two and the last photo don't look like the same contam

and are those polypropylene cups? can you describe how your process?

what kind of modifications, cook times, agar-recipe, etc. are you using on the cups?


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OfflineGiermmo
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Re: Help with Contam Identification [Re: MonkeyJesusFresco]
    #28178578 - 02/09/23 12:20 AM (1 year, 11 months ago)

How do you pour you plates, do you use SAB?


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OfflineBigSquid
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Re: Help with Contam Identification [Re: MonkeyJesusFresco]
    #28178598 - 02/09/23 12:43 AM (1 year, 11 months ago)

Sure!

They're 2oz polypropylene cups.

The agar recipe I use is 12g Potato Flakes, 8grams Telephone Agar Agar, 4 oz karo, 1 drop gel food coloring in blue. Mixed with 500ml boiling water in a round media pour bottle ((--Karter Scientific brand)).

((I typically skip mixing it together in a saucepan and opt for using my electric kettle to boil the water to pour directly into the media bottle and shaking the hell out of it until everything has dissolved. Mainly because my kettle has only ever had water in it and my saucepans...have not, second is because I dont want to have another fucking dish to wash. The measuring tools and media bottles I use for cultivation ONLY get used for cultivation and nothing else. ))

I use an Instapot to PC, and adjust for the pressure difference by an extra 15 minutes, for agar I usually do around 75 minutes-- so far I haven't seen any caramelization, I haven't had a consistent problem with weird shit cropping up, but, if I need leave it in longer please let me know. 

I take it out of the instapot after about 20 minutes after the cycle is over, give it a swirl, and set it to start cooling. It's typically around 180° at this point.

I don't have a flow hood and my hands are too shakey for me to trust this shit around my arms in my SAB -- so I use the Oven Tek, 250° cracked open, with a mesh strainer hanging over I set my cups and lids on.

Wait for it to cool to 150° -- pour pour pour pour pour pour, snap the lids on, stack them, and move them to the storage bin I keep them in. I don't modify the lids.

I usually wait until half a day or so to start using them, typically it's been making them at night and then the next morning doing transfers or innoculations -- I've been trying to use them before they hit more than a few weeks old. I use my SAB for transfers, innoculating, etc.




-- I took them out and gave them a good look over, and, we've got a couple of different things going on other than the weird dehydrated urine yellow spots and the creeping slime.

I did my best with the photos:



I pulled both bags from the first botched attempt ((which was the 17th, and not the 11th)) -- all cubes
'A' Labeled cups had the worst of the yellowing/creeping side contam ((I think they mss was Pink Buff)) and is the one I'm having the most trouble with.

'B' had some other funky stuff going on, but had a lot more cube-ish looking mycellium / growth -- most of the contam for 'B' has a lot of pink coloring-- I think it's lipstick, the jar I also used the MSS had the same in the bottom.

The unlabelled one that has a creeper in the side looks how 'A' did when I first noticed the side looked off, it's paler and more white, though.

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OfflineGiermmo
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Re: Help with Contam Identification [Re: BigSquid] * 1
    #28178632 - 02/09/23 01:23 AM (1 year, 11 months ago)

Oven tek is outdated bullshit, use sab and spray everything with iso (except inside of the cups OFC).
I am shaky too, needs practice. I think that either you got contams from "oven tek" or your cups are dirty, do you use gloves? Do you spray them with iso?
It seems like bacterial contam to me but it's BELOW the agar surface, so i think your contamination is there even before inoculation.
If it was from inoculation, it would probably stay on top of agar.


--------------------
Don't worry mate, what you're gonna feel is a slight burning in your heart, your lungs, your brain, your kidneys, your liver, bottom of your feet, your eyeballs, then you start bleeding from your nose and ears, then you start tripping out and seeing everything in the form of an anime cartoon which is really fucking cool.

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Offlinehazyhorse
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Re: Help with Contam Identification [Re: Giermmo]
    #28178645 - 02/09/23 01:38 AM (1 year, 11 months ago)

:whathesaid:

it's 100% from trying to use your oven as a flowhood. SAB is the bare minimum for doing agar work imo. you don't even really need to spray the SAB or anything, a ton of people use them dry. as long as there aren't any drafts in your room and you don't have AC or fans running you should be golden. i'd recommend trying containers like these or screw top pp5 containers and doing no pour agar. one less contamination vector to worry about and one less thing to fuck up along the way. they come out of the PC sterile and will remain that way until you decide to use them, no pouring and leaving a a chance for your own human error there


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Offlinefiddle_head
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Re: Help with Contam Identification [Re: BigSquid]
    #28178646 - 02/09/23 01:39 AM (1 year, 11 months ago)

There's the same 'image deleted' pop-up when you click that's been happening everywhere.


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OfflineGiermmo
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Re: Help with Contam Identification [Re: hazyhorse]
    #28178668 - 02/09/23 02:18 AM (1 year, 11 months ago)

Quote:

hazyhorse said:
:whathesaid:

it's 100% from trying to use your oven as a flowhood. SAB is the bare minimum for doing agar work imo. you don't even really need to spray the SAB or anything, a ton of people use them dry. as long as there aren't any drafts in your room and you don't have AC or fans running you should be golden. i'd recommend trying containers like these or screw top pp5 containers and doing no pour agar. one less contamination vector to worry about and one less thing to fuck up along the way. they come out of the PC sterile and will remain that way until you decide to use them, no pouring and leaving a a chance for your own human error there




Yeah i'm also not spraying the inside, i did mean spray things that were exposed to air outside of SAB and aren't sterilised.
Also gloves every time i do something outside of SAB with them (Flaming scalpel etc.)
Yeah no pour could help, try that and report back :smile:


--------------------
Don't worry mate, what you're gonna feel is a slight burning in your heart, your lungs, your brain, your kidneys, your liver, bottom of your feet, your eyeballs, then you start bleeding from your nose and ears, then you start tripping out and seeing everything in the form of an anime cartoon which is really fucking cool.

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InvisibleCommanderDemander
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Re: Help with Contam Identification [Re: Giermmo]
    #28178905 - 02/09/23 07:04 AM (1 year, 11 months ago)

I use 2oz condiment cups as agar plates and pour in a SAB. Practice makes perfect but out of 400 poured plates I have only had 2 contam from poor pours. I keep my cups for at least a week before use to ensure no contam and I have had no issues with the plates drying up. At least condiment cups are cheap so if you have issues practicing it isn't a large financial concern.

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OfflineBigSquid
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Re: Help with Contam Identification [Re: Giermmo]
    #28179564 - 02/09/23 01:59 PM (1 year, 11 months ago)

I'll check it out and try a no pour! 

I'm also just gonna bite the bullet and invest in a larger SAB to do my pours in -- the one I made is on the smaller size due to space issues. Smaller work space and shakey hands just seemed like it was begging for an accident. Might also make everything else a bit easier, too, and I can work with more at a time. 

So far I've done probably poured 50ish cups or so -- and I've used about 40 of those. Aside from the first 10 or so that got wrecked ((seen in the photos -- theyre the ones with the yellow/greenish tint in the blue)), I've only had maybe 5 that had *visible* contamination -- and 3 of those were after I did transfers/innocs ((One of these is seen in the first photo)). The other two I had been storing-- one had a spot of bacteria and the other had a mold spot.

I'll keep an eye on my other plates I've used and watch the batch I just poured a few nights ago in the meantime.

I'm gonna attribute the lack of *current* Visible contam to the fact I bleach the hell out of my work spaces before and after I do work and run an air purifier in my apartment ((--not when I'm working, just in general for a few hours a day)) -- also probably a stupid amount of dumb luck. Also if this isn't advisable please let me know!

I do use gloves and iso them and my pour bottle frequently while working -- I don't use it on my counter tops or workspace that got bleach wiped-- I know they shouldn't be mixed.

Thanks for the advice, everyone!

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Invisibledowodenum
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Re: Help with Contam Identification [Re: BigSquid]
    #28179583 - 02/09/23 02:10 PM (1 year, 11 months ago)

Instant pot? They are 11.5 psi IIRC, so I'd definitely go a bit longer than 20 minutes.


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OfflineBigSquid
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Re: Help with Contam Identification [Re: dowodenum]
    #28179683 - 02/09/23 03:28 PM (1 year, 11 months ago)

I read somewhere to add extra time for each level of pressure difference when using an Insta-- I'll hunt down the thread I checked out for that one to double check, tho. I've been doing an extra 15 minutes for each level, so my time typically runs an extra 45 minutes -- for agar I've been doing 75 minutes. :shrug: I know carmelizing is a concern, but, so far I haven't experienced it and it's been pouring well.

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Invisibledowodenum
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Re: Help with Contam Identification [Re: BigSquid]
    #28179917 - 02/09/23 06:08 PM (1 year, 11 months ago)



from https://www.shroomery.org/forums/showflat.php/Number/19342794 but that's an oold thread.

SPG has a more recent one but it's assuming strictly 15 PSI: https://www.shroomery.org/forums/showflat.php/Number/28166056

Not sure how either would scale down to sauce cups. Maybe start at an hour and scale back until your contam rate is unacceptable? Of course your sterile technique will probable improve in the meanwhile, so it won't be a definitive test.


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