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Offlinesmokedtrolli
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9/9 oat jars contam'd with what appears to be Penicillium. Why?
    #26431456 - 01/13/20 02:20 PM (4 years, 16 days ago)

I inoculated these jars with agar wedges that were made about a month ago. The plate I used for these showed very strong rhizomorphic growth with complete uniformity. There was no suspicion of other kinds of growth on the plate that wasn't mycelium.

I prepped the oats by boiling for ~40 minutes, strained them in a colander, and then laying them out on paper towels to dry them as much as possible before adding them to the jars and PCing. I PCed at 15 PSI for 90 minutes and didn't touch them until the following day.

When I removed them, there were no signs of wetness. I inoculated using a SAB, and x-acto knife that was heated via butane torch in between each jar. I wore gloves (though, my forearms were exposed) and tried to avoid moving anything that was not sterile above the jar openings or the agar plate during the process. As I was dropping the wedge into the jars, I did my best to only remove the jar's lid for as long as it took me to get it off the knife.

Afterward, the jars were kept in my basement at low temps (60s I'm assuming). I waited for some mycelium to grow off onto local grains surrounding the wedges before I shook them. After the shake is when the same kind of mold appeared in every jar. Bluish-green, somewhat fuzzy. I'm 99.99% sure it is not bruising.

What are the theories here? Did the plate have covert growth? Did the grains pick up something from the paper towels/colanders? Is my sterile technique dogshit? Or just absolutely horrendous luck?


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: smokedtrolli]
    #26431464 - 01/13/20 02:25 PM (4 years, 16 days ago)

It could be any number of things... improper sterilization of grains, sloppy SAB work or dirty plates... if they are all contaminated I'd probably lean towards a dirty plate...


U got any pics?


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Offlinesmokedtrolli
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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: SpunkyMonkey88]
    #26431470 - 01/13/20 02:28 PM (4 years, 16 days ago)

Yeah, here are pics:







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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: smokedtrolli]
    #26431477 - 01/13/20 02:32 PM (4 years, 16 days ago)

That spawn is contaminated for sure but do u have any pics of your agar


Edited by SpunkyMonkey88 (01/13/20 02:33 PM)


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Offlinesmokedtrolli
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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: SpunkyMonkey88]
    #26431479 - 01/13/20 02:32 PM (4 years, 16 days ago)

Quote:

SpunkyMonkey88 said:
That spawn is contaminated




As in, the plate was?


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: smokedtrolli]
    #26431482 - 01/13/20 02:34 PM (4 years, 16 days ago)

Quote:

smokedtrolli said:
Quote:

SpunkyMonkey88 said:
That spawn is contaminated




As in, the plate was?



I would think so I dont see any healthy mushroom mycelium I'm thinking you inoculated your jars with mold


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: SpunkyMonkey88]
    #26431485 - 01/13/20 02:35 PM (4 years, 16 days ago)

Either that or you didnt PC your jars long enough, most people go two hours...

I would think 100% moldy jars either means you inoculated with mold or your grains wernt sterile. I'm thinking your plate wasn't even mushrooms mycelium itd help if u had some pics


Edited by SpunkyMonkey88 (01/13/20 02:38 PM)


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Invisiblerickyswamps
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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: SpunkyMonkey88]
    #26431509 - 01/13/20 02:43 PM (4 years, 16 days ago)

PC cycles always kill mold.  Its probably from the plates if it was 9 out of 9.  If they look perfect, then it is from SAB technique.


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Offlinesmokedtrolli
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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: SpunkyMonkey88]
    #26431513 - 01/13/20 02:44 PM (4 years, 16 days ago)

Quote:

SpunkyMonkey88 said:
Either that or you didnt PC your jars long enough, most people go two hours...

I would think 100% moldy jars either means you inoculated with mold or your grains wernt sterile. I'm thinking your plate wasn't even mushrooms mycelium itd help if u had some pics





This is what's leftover. The middle of it contains a piece from a previous plate.



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Edited by smokedtrolli (01/13/20 02:59 PM)


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: smokedtrolli]
    #26431580 - 01/13/20 03:13 PM (4 years, 16 days ago)

Your two options are you didn't notice your plate wasn't 100% healthy.
Or it got in during the transfer/inoculation


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Offlinesmokedtrolli
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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: bodhisatta]
    #26431607 - 01/13/20 03:23 PM (4 years, 16 days ago)

Quote:

bodhisatta said:
Your two options are you didn't notice your plate wasn't 100% healthy.
Or it got in during the transfer/inoculation




That's what I figured. Probably during transfer if I had to guess.


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: smokedtrolli]
    #26431608 - 01/13/20 03:23 PM (4 years, 16 days ago)

Or your gas exchange filters are insufficient or incorrectly made


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: bodhisatta]
    #26431663 - 01/13/20 03:47 PM (4 years, 16 days ago)

Quote:

bodhisatta said:
Or your gas exchange filters are insufficient or incorrectly made




Ya those are possibilities I didnt think of... It doesnt look like whatever is in your jars was on your plate but who knows they are way overgrown but look good to me....


I'm gonna say you didnt PC properly after seeing ur leftover plate...but it's all just an educated guess at this point...

You'd have to really have some shitty technique in the SAB to introduce contamination 9 out of 9 times in between the plate and the jar(s)


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: bodhisatta]
    #26431673 - 01/13/20 03:49 PM (4 years, 16 days ago)

Quote:

bodhisatta said:
Or your gas exchange filters are insufficient or incorrectly made




On the jars? They're 1/4in. holes stuffed with polyfill. Is that alright or should I use tyvek filters instead?


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: smokedtrolli]
    #26431675 - 01/13/20 03:50 PM (4 years, 16 days ago)

Poly is fine. If it wasn't stuffed tight enough that could be your problem though


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: SpunkyMonkey88]
    #26431676 - 01/13/20 03:51 PM (4 years, 16 days ago)

I mean, I suppose I could have fucked up PCing, but that's so incredibly simple I don't see how anyone could fuck it up unless they were way below PSI or misjudged time by an hour.


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: bodhisatta]
    #26431682 - 01/13/20 03:53 PM (4 years, 16 days ago)

Hmm... I didn't quite grab the biggest ball of polyfill when I made those lids... I suppose it could have been somewhat loose.


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: smokedtrolli]
    #26431688 - 01/13/20 03:59 PM (4 years, 16 days ago)

Well if it was from the agar/lid/inoculation it would have started from the top of the jar... if it were the grains itd be sporadic throughout the entire jar...

Where did you first see the contamination take hold? Any kind of pattern? That may help you figure out the origin


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: SpunkyMonkey88]
    #26431691 - 01/13/20 04:01 PM (4 years, 16 days ago)

That is assuming you didnt shake your jar after dropping the agar...

I always like to drop it and let it sit for a while to see what developers before shaking it that way you know what is going on and what you're spreading VIA shaking...


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: SpunkyMonkey88]
    #26431693 - 01/13/20 04:03 PM (4 years, 16 days ago)

I shook most of them after not seeing any mold and having some mycelium growth off of the wedge. Of the ones I did not shake, the contamination actually did originate from the top.


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: smokedtrolli]
    #26431702 - 01/13/20 04:09 PM (4 years, 16 days ago)

What kind of lids?  pics?


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: smokedtrolli]
    #26431704 - 01/13/20 04:12 PM (4 years, 16 days ago)

Quote:

smokedtrolli said:
I shook most of them after not seeing any mold and having some mycelium growth off of the wedge. Of the ones I did not shake, the contamination actually did originate from the top.




If you didn't see any growth (mushroom or otherwise) from anywhere but the top then you could probably forget the PC theory I suggested and assume it was from the lids or the agar or sloppy innlocation technique...

All u can do at this point is take some transfers from that slice u have left and tighten up your technique when you make your second attempt...

I think this is super weird... I've never seen 100% contamination rate before except for dumb shit like open air inoculated grain jars or something crazy like that..


I'm also very new to this as well so....


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: SpunkyMonkey88]
    #26431762 - 01/13/20 04:56 PM (4 years, 16 days ago)

How old are those jars and how long after the shake?
That’s an impressive amount of penicillium!


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Offlinesmokedtrolli
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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: StygianKnight]
    #26432951 - 01/14/20 10:37 AM (4 years, 15 days ago)

Quote:

StygianKnight said:
How old are those jars and how long after the shake?
That’s an impressive amount of penicillium!




2-3 days after the shake, the jars are probably a week old. Pretty aggressive stuff.


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Offlinesmokedtrolli
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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: rickyswamps]
    #26432954 - 01/14/20 10:39 AM (4 years, 15 days ago)

Quote:

rickyswamps said:
What kind of lids?  pics?




1/4inch holes stuffed with polyfill. I'm not sure if that necessitates a picture or not considering half of the people who have grown w/ grains do the same thing. Nothing really special.


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: smokedtrolli]
    #26432958 - 01/14/20 10:41 AM (4 years, 15 days ago)

Quote:

smokedtrolli said:
Quote:

rickyswamps said:
What kind of lids?  pics?




1/4inch holes stuffed with polyfill. I'm not sure if that necessitates a picture or not considering half of the people who have grown w/ grains do the same thing. Nothing really special.




Ya that's pretty hard to fuk up 9/9times... weird dude.

I cant process how this is possible unless you licked your scalpel between jars or something


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: SpunkyMonkey88]
    #26432960 - 01/14/20 10:44 AM (4 years, 15 days ago)

Quote:

SpunkyMonkey88 said:
Quote:

smokedtrolli said:
Quote:

rickyswamps said:
What kind of lids?  pics?




1/4inch holes stuffed with polyfill. I'm not sure if that necessitates a picture or not considering half of the people who have grown w/ grains do the same thing. Nothing really special.




Ya that's pretty hard to fuk up 9/9times... weird dude.

I cant process how this is possible unless you licked your scalpel between jars or something




I think it's my SAB actually. The bottom lacks uniformity so it isn't flush with the table, leaving space for air to enter under the SAB. That's my only guess. The colonized agar plate was left long enough for anything that wasn't mycelium to develop and reveal itself.

Probably gonna go buy a new container and make another SAB. And just be more rigorous with cleaning the work area before hand too.


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: smokedtrolli]
    #26432970 - 01/14/20 10:53 AM (4 years, 15 days ago)

Ya dude that sounds like a good plan. My table isn't uniformly flat either. It's made of 3 1' wide boards with a 1/4" gap in between them. So I lay down a big piece of plastic then a layer of paper toweling then i spray it down...

I've worked with just under 100 agar plates always pouring more than I need so they always sit for a week before using and I've only ever seen 1 plate contaminate before using it...

Just how I do shit and it works well for me...


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: smokedtrolli]
    #26432975 - 01/14/20 10:56 AM (4 years, 15 days ago)

I have no real knowledge about this but it seems likely it has something to do with the grains. Maybe the jars weren’t sterile?

If the agar was good that and the grains are really the only common denominators. I can’t imagine it being the SAB, it seems like you could do it open air and not get that much/consistent contamination.

Definitely weird.


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: A.k.a]
    #26432984 - 01/14/20 11:01 AM (4 years, 15 days ago)

Quote:

A.k.a said:
I have no real knowledge about this but it seems likely it has something to do with the grains. Maybe the jars weren’t sterile?

If the agar was good that and the grains are really the only common denominators. I can’t imagine it being the SAB, it seems like you could do it open air and not get that much/consistent contamination.

Definitely weird.




That's what I was saying at first but dude seems pretty confident he didnt fuk up the PC cycle...

He said he ran it for 1.5 hrs I always go for 2 so I dont know if that 30 mins could of left that contaminate in every jar or not...


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: SpunkyMonkey88]
    #26433017 - 01/14/20 11:45 AM (4 years, 15 days ago)

Quote:

SpunkyMonkey88 said:
Quote:

smokedtrolli said:
Quote:

rickyswamps said:
What kind of lids?  pics?




1/4inch holes stuffed with polyfill. I'm not sure if that necessitates a picture or not considering half of the people who have grown w/ grains do the same thing. Nothing really special.




Ya that's pretty hard to fuk up 9/9times... weird dude.

I cant process how this is possible unless you licked your scalpel between jars or something



Even licking the scalpel couldn't cause this. More likely that would cause a pretty bad case of bacteria.

OP 1.5 hours ain't bad but for oats I'd definitely increase to 2 hours and that also depends on your elevation.
Also I didn't see anyone ask did you vent your PC before building pressure? This is important.
I'd do another run. Stuff polyfil real tight, vent pc, and sterilize for 2 hours this time. Use the same culture but transfer to a new plate and post pics here so we can try to help you identify if anything is hiding in there.

With any luck you will have clean spawn in no time.


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: gizmo1]
    #26433095 - 01/14/20 12:53 PM (4 years, 15 days ago)

Quote:

gizmo1 said:
Also I didn't see anyone ask did you vent your PC before building pressure? This is important.





Considering I don't know what you mean by "vent", no, lol. Oops? I usually just everthing clean w/ bleach and soap before starting over again.


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: smokedtrolli]
    #26433109 - 01/14/20 01:01 PM (4 years, 15 days ago)

He means putting the jars in the PC and the lid on the PC but leaving the "jiggler/rocker" off while the steam vents out for 10 mins or so... they say that it is pretty important
.. I always do for that reason but I've also forgot and used to not vent until reading that with out any notable contaminations...


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: SpunkyMonkey88]
    #26433127 - 01/14/20 01:09 PM (4 years, 15 days ago)

Quote:

SpunkyMonkey88 said:
He means putting the jars in the PC and the lid on the PC but leaving the "jiggler/rocker" off while the steam vents out for 10 mins or so... they say that it is pretty important
.. I always do for that reason but I've also forgot and used to not vent until reading that with out any notable contaminations...



This just means you weren't pcing as long as you thought you were well not at temp anyways. If using a presto steam still escapes so you will eventually get the temps up but if you are only pcing for a hour and a half without venting then who knows how long you were actually at sterilization temps. You want only steam in the pc not steam and air pockets. The temperature of the air pockets will be much lower than the temperature of the steam. Always vent your PC.


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: gizmo1]
    #26433130 - 01/14/20 01:11 PM (4 years, 15 days ago)

Quote:

gizmo1 said:
Quote:

SpunkyMonkey88 said:
He means putting the jars in the PC and the lid on the PC but leaving the "jiggler/rocker" off while the steam vents out for 10 mins or so... they say that it is pretty important
.. I always do for that reason but I've also forgot and used to not vent until reading that with out any notable contaminations...



This just means you weren't pcing as long as you thought you were well not at temp anyways. If using a presto steam still escapes so you will eventually get the temps up but if you are only pcing for a hour and a half without venting then who knows how long you were actually at sterilization temps. You want only steam in the pc not steam and air pockets. The temperature of the air pockets will be much lower than the temperature of the steam. Always vent your PC.




Do you think that was the source of my penicillium tragedy?


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: smokedtrolli]
    #26433144 - 01/14/20 01:16 PM (4 years, 15 days ago)

Quote:

smokedtrolli said:
Quote:

gizmo1 said:
Quote:

SpunkyMonkey88 said:
He means putting the jars in the PC and the lid on the PC but leaving the "jiggler/rocker" off while the steam vents out for 10 mins or so... they say that it is pretty important
.. I always do for that reason but I've also forgot and used to not vent until reading that with out any notable contaminations...



This just means you weren't pcing as long as you thought you were well not at temp anyways. If using a presto steam still escapes so you will eventually get the temps up but if you are only pcing for a hour and a half without venting then who knows how long you were actually at sterilization temps. You want only steam in the pc not steam and air pockets. The temperature of the air pockets will be much lower than the temperature of the steam. Always vent your PC.




Do you think that was the source of my penicillium tragedy?



Can't say with 100% certainty but it could be. Not venting combined with low sterilization times could very well do it.


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: gizmo1]
    #26433173 - 01/14/20 01:24 PM (4 years, 15 days ago)

Sounds likely.

Venting I always thought you let it run until a solid cone of steam was coming out.


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: A.k.a]
    #26433237 - 01/14/20 01:53 PM (4 years, 15 days ago)

So a pressure cooker that wasn't vented but still reads X-psi is actually cooler than the same PC vented at the same PSI?


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OfflineCamera93
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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: SpunkyMonkey88]
    #26433251 - 01/14/20 01:57 PM (4 years, 15 days ago)

Quote:

SpunkyMonkey88 said:
So a pressure cooker that wasn't vented but still reads X-psi is actually cooler than the same PC vented at the same PSI?




A write up from Bod

The Importance of Venting a Pressure Cooker or Autoclave

BOD's Easy AF how to use your pressure cooker for mycology TEK


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: Camera93]
    #26433322 - 01/14/20 02:24 PM (4 years, 15 days ago)

Quote:

Camera93 said:
Quote:

SpunkyMonkey88 said:
So a pressure cooker that wasn't vented but still reads X-psi is actually cooler than the same PC vented at the same PSI?




A write up from Bod

The Importance of Venting a Pressure Cooker or Autoclave

BOD's Easy AF how to use your pressure cooker for mycology TEK




Seems like an interesting read I'm gonna check it out when I'm off work...


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: SpunkyMonkey88]
    #26433363 - 01/14/20 02:43 PM (4 years, 15 days ago)

It's not your pc cycle . Molds are killed off even with pastirinzing temps  so unless you just sat them in hot tap water that's not your issue.
Your issue is inoculant , if it was your filters you would see it start at the top of your grain and would likely have more than one type of contaminate , a shitty technique you may lose half your jars but get lucky with the other half, the common factor here is your inoculant


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: gizmo1]
    #26433413 - 01/14/20 03:06 PM (4 years, 15 days ago)

Quote:

gizmo1 said:




Can't say with 100% certainty but it could be. Not venting combined with low sterilization times could very well do it.




Can we reiterate the discussion of sterility during inoculation? My understanding was that that is extremely important yet a few of you are saying you can get away with open-air inoculation? I'm confused.


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: cronicr]
    #26433416 - 01/14/20 03:09 PM (4 years, 15 days ago)

Quote:

cronicr said:
It's not your pc cycle . Molds are killed off even with pastirinzing temps  so unless you just sat them in hot tap water that's not your issue.
Your issue is inoculant , if it was your filters you would see it start at the top of your grain and would likely have more than one type of contaminate , a shitty technique you may lose half your jars but get lucky with the other half, the common factor here is your inoculant




So basically there was mold on that plate that I didn't notice. I'm willing to believe that I guess; perhaps it was premature or something and hadn't yet released spores?


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: smokedtrolli]
    #26433427 - 01/14/20 03:12 PM (4 years, 15 days ago)

If you're using needles and injection ports you can do inoculation open air


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: smokedtrolli]
    #26433428 - 01/14/20 03:13 PM (4 years, 15 days ago)

Yeah there are molds that don't tend to sporulate till they get fresh air, more nutrition, broken colony ECT, best bet is to start fresh with new filters and post pics of your cultures before going to grain


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: cronicr]
    #26433433 - 01/14/20 03:15 PM (4 years, 15 days ago)

Quote:

cronicr said:
Yeah there are molds that don't tend to sporulate till they get fresh air, more nutrition, broken colony ECT, best bet is to start fresh with new filters and post pics of your cultures before going to grain




I have two other plates that are colonized but the lid is covered with condensation from the inside; supplying a clear picture would be difficult. Use or discard?


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: smokedtrolli]
    #26433436 - 01/14/20 03:16 PM (4 years, 15 days ago)

Make transfers and take pics of the donor plate afterward


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: cronicr]
    #26433441 - 01/14/20 03:19 PM (4 years, 15 days ago)

You can put something warm on the plate to clear up condensation real quick too if you want. Sounds like a lot of people use a cup of hot water


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: cronicr]
    #26433442 - 01/14/20 03:19 PM (4 years, 15 days ago)

Quote:

cronicr said:
Make transfers and take pics of the donor plate afterward




Gotcha. Unrelated: what's the max amount of time sterilized grain should be kept without being inoculated? Just prepped a bunch of grain that I'd rather not waste.


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Re: 9/9 oat jars contam'd with what appears to be Penicillium. Why? [Re: smokedtrolli]
    #26436495 - 01/16/20 10:04 AM (4 years, 13 days ago)

Quote:

smokedtrolli said:
Quote:

cronicr said:
Make transfers and take pics of the donor plate afterward




Gotcha. Unrelated: what's the max amount of time sterilized grain should be kept without being inoculated? Just prepped a bunch of grain that I'd rather not waste.




Of its properly sterilized, many months
.. I would think until it dries out or the structure of the grains start to degrade...

I've used jars that were 3 months old with no problems whatsoever


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