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Prinz Eugen
Prinz


Registered: 12/02/23
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Loc: Austria
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Re: Recognizing and dealing with contamination [Re: LadysKnight]
#28574305 - 12/08/23 11:59 AM (1 month, 19 days ago) |
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What am i growing here? Some Aspergillus spp.? Never had this beautiful mold before.
-------------------- Seeking Spores of: P. Serbica, P. Medullosa, P. Crobula, P. Pelliculosa PM me!
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Zwinst
Stranger


Registered: 02/27/21
Posts: 126
Loc: Germany
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Re: Recognizing and dealing with contamination [Re: Bigworm]
#28574407 - 12/08/23 01:24 PM (1 month, 19 days ago) |
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Ähm, looks like maybe the proper myceQuote:
Bigworm said: I may have found another vector for contamination. It's not a big vector and I haven't seen anyone mention it but I noticed it. While prepping grain I found some dry grain getting stuck around the rim of my strainer from mixing it up while rinsing the grain. I use this type of strainer
 And here is the lip in question.
 I thought about it and when I dump my properly hydrated grain into the strainer a few dry grains my be getting released from the rim into them. It's probably never a lot, but I'm assuming a few dozen dry grains getting into the prepped grain might be carrying endospores. Being dry they might not get fully hydrated by the prepped grain during the PC cycle and even if they do, I suspect they aren't getting sterilized properly. I'm not sure this is 100% correct just an assumption and another place to look for a possible contamination point.
I used to use the exact same strainer and i switched to something like this:

Combined with the buckets i use they make a pretty good fit. It makes it way more easy to properly wash the grain.
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Smellyhobbit
Actual Retard



Registered: 04/01/22
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Re: Recognizing and dealing with contamination [Re: Zwinst]
#28575886 - 12/09/23 01:53 PM (1 month, 18 days ago) |
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Hey contam fam. Big question for you.
What would cause all my A2G inoculations to succumb to the same mold across 10 cultures, 4 grain preps, and 3 SAB sessions when LC works perfectly even when made from some of those same cultures that fail on grain?
I’m losing a ton of jars here at the end of the year. All the same contam. If LC works, I’m assuming it isn’t a lid or filter failure. And the cultures I’m using to make said LC are clean on the plate and in the broth, but fail on grain.
What could I be doing wrong?
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PBJ710
Strangler


Registered: 07/05/19
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Re: Recognizing and dealing with contamination [Re: Smellyhobbit]
#28575903 - 12/09/23 02:11 PM (1 month, 18 days ago) |
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The only thing I can think of that might be different in the working and failing ones would possibly be sterile technique during inoculation. Assuming this is FFU based clean work, have you tested your filter recently with a blank petri to see if anything grows on it after a nominal exposure to what should be clean air? Are you just doing a sneaky transfer with the lid held by the same hand as the petri or are you taking the lid off completely and setting it down somewhere in the mean time?
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Smellyhobbit
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Re: Recognizing and dealing with contamination [Re: PBJ710]
#28575915 - 12/09/23 02:16 PM (1 month, 18 days ago) |
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SAB actually. I haven’t like changed my technique at all except I’ve been more careful of giving the SAB time to settle and surface cleaning. Hadn’t helped, unfortunately
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Baba Yaga
♥ coir grower

Registered: 09/13/20
Posts: 3,955
Loc: Hyperspace Chicken Coop
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Re: Recognizing and dealing with contamination [Re: Smellyhobbit]
#28575938 - 12/09/23 02:34 PM (1 month, 18 days ago) |
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Wait can you clarify please? You mean LC does contaminate as well once put to grain or do LC inoculations work without a problem and it's just agar to grain that tams out?
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Smellyhobbit
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Re: Recognizing and dealing with contamination [Re: Baba Yaga]
#28575951 - 12/09/23 02:50 PM (1 month, 18 days ago) |
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Quote:
Baba Yaga said: Wait can you clarify please? You mean LC does contaminate as well once put to grain or do LC inoculations work without a problem and it's just agar to grain that tams out?
LC has a 100% success rate A2A performs exactly as it should A2LC Broth has a 100% success rate A2G went from optimal success to a 100% failure rate without warning and all at once.
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Smellyhobbit
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Re: Recognizing and dealing with contamination [Re: Smellyhobbit]
#28575952 - 12/09/23 02:52 PM (1 month, 18 days ago) |
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Also, the same agar wedges I’m using to inoculate my clean LC fail on grain.
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Baba Yaga
♥ coir grower

Registered: 09/13/20
Posts: 3,955
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Re: Recognizing and dealing with contamination [Re: Smellyhobbit] 1
#28575992 - 12/09/23 03:26 PM (1 month, 18 days ago) |
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Mmm very mysterious. The different combinations seem to cancel each other out as the cause for contam.
Grain works with everything apart from agar.
Agar works with everything apart from grain.
So if the different media do work fine except from this particular combination and coincidence can be ruled out then it must be something else....well, most likely. Have you changed anything else in the process in recent past or do you use different tools for the task? I assume you have not recently got into the habit of wearing a flamboyant cat fur coat on A2G days?
I'll keep thinking about this.
Sorry for your losses.
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Smellyhobbit
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Re: Recognizing and dealing with contamination [Re: Baba Yaga] 1
#28576000 - 12/09/23 03:28 PM (1 month, 18 days ago) |
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Wait it could be my flamboyant pink fur coat?
Nah but everything is the same. Last inoculations in September were all fine. Everything from the start of October to now that’s been A2G has failed. No new tools, no novel changes besides the way I prep my WBS. But it worked once extremely well and hasn’t since.
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Zwinst
Stranger


Registered: 02/27/21
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Re: Recognizing and dealing with contamination [Re: Smellyhobbit]
#28576008 - 12/09/23 03:40 PM (1 month, 18 days ago) |
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Maybe something went wrong with your grain sterilisation? I guess your grain doesn´t sit idle for weeks before inocculation... Maybe endospores. Do you pasteurize your grain before sterilisation? Helps a lot with endospores, thats why i soak my grain in boiling water at least 24h before sterilisation.
Did you properly clean your grain containers from the outside before opening? You could have dragged something in, theoretically.
What exactly is your contaminant?
This is a interesting mystery.
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Baba Yaga
♥ coir grower

Registered: 09/13/20
Posts: 3,955
Loc: Hyperspace Chicken Coop
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Re: Recognizing and dealing with contamination [Re: Smellyhobbit]
#28576011 - 12/09/23 03:44 PM (1 month, 18 days ago) |
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Oh fuck, that is a long time.
Not doing so well either at the moment which is why I popped by. Half the stuff that tams out I expect to do so because of science but the half I really like to get working is not doing great except some success with subtrops. I think the soil on my property is heavily infested with fusarium which seems to cause me problems during a certain time in the year when it's dryer and windy. A few things are pointing at this including some plants in the garden.
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Smellyhobbit
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Re: Recognizing and dealing with contamination [Re: Zwinst]
#28576025 - 12/09/23 04:04 PM (1 month, 18 days ago) |
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Quote:
Zwinst said: Maybe something went wrong with your grain sterilisation? I guess your grain doesn´t sit idle for weeks before inocculation... Maybe endospores. Do you pasteurize your grain before sterilisation? Helps a lot with endospores, thats why i soak my grain in boiling water at least 24h before sterilisation.
Did you properly clean your grain containers from the outside before opening? You could have dragged something in, theoretically.
What exactly is your contaminant?
This is a interesting mystery.
THIS is how I prep my grain.
It can sit idle up to maybe a week, but that’s never been an issue. I also wiped down these last jars with an iso towel, not that that makes them sterile or anything, but I figured it might help idk. It’s also not an issue with my LC spawn.
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Zwinst
Stranger


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Re: Recognizing and dealing with contamination [Re: Smellyhobbit] 1
#28576082 - 12/09/23 04:37 PM (1 month, 18 days ago) |
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Alright, you sterilise pretty soon after saoking. With endospores the idea is to crack them with heat and give them time to germinate. 24h usually is enough. I always wait till the next day. Sometimes a day more (not by choice). I drain and wash the grain thoroughly to get rid of excess yeast and starch (i use a mix of a lot of grains, they are sticky), let it drain properly and sterilise.
Haven´t had any mystery contaminations -inside my jars- ever since.
But you say lc to grain is fine.
I don´t get whats going wrong. You are probably using the same tools to transfer your agar wedges you use for everything else. It is happening in the same space. With the same preparation. Beats me.
Well, if you rule out the impossible, anything that remains, however improbable... It IS your fur coat!
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Way
The


Registered: 01/14/23
Posts: 4,336
Loc: A long way away
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Re: Recognizing and dealing with contamination [Re: Zwinst]
#28576154 - 12/09/23 05:22 PM (1 month, 18 days ago) |
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Your sterile work is usually spot on. You're not a noob and you know what you're doing. A2A and A2LC working without failure shows your transfers are fine.
You are running multiple different cultures so it can't be a culture related issue.
That leaves grain prep and sterilization right?
Have you left a control jar running without inoculating to see if it succumbs as well? If it does, it is definitely the PC giving up the ghost or an issue with the grain prep. If it doesn't, you've started doing something different during A2G that is causing the issue I would assume.
--------------------
That's the way she goes, boys. Sometimes she goes, sometimes she doesn't, cause that's the fuckin way she goes.
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rumfor69
Bodhicitta Cultivator



Registered: 08/05/11
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Re: Recognizing and dealing with contamination [Re: Way]
#28576198 - 12/09/23 06:04 PM (1 month, 18 days ago) |
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Only thing I can think of is the technique when doing g2g in an sab. There's a lot more hovering over the receiver jar while the cement mixer movement is happening. It's a lot different than the clean outreach movement of a scalpel or syringe. I had lots of mold issues with g2g in an sab back in the day and only a flowhood solved it.
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LadyBird123


Registered: 09/27/22
Posts: 257
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Re: Recognizing and dealing with contamination [Re: rumfor69]
#28576448 - 12/09/23 10:06 PM (1 month, 18 days ago) |
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 Agar to grain aswell… compounded with slower colonization times. Turns into a competition.
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hazyhorse
scoobin



Registered: 03/19/19
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Re: Recognizing and dealing with contamination [Re: Smellyhobbit]
#28576524 - 12/10/23 12:59 AM (1 month, 18 days ago) |
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Quote:
Smellyhobbit said: Wait it could be my flamboyant pink fur coat?
Nah but everything is the same. Last inoculations in September were all fine. Everything from the start of October to now that’s been A2G has failed. No new tools, no novel changes besides the way I prep my WBS. But it worked once extremely well and hasn’t since.
have you been baking your verm lately though ?? just a thought 
Quote:
Baba Yaga said: Oh fuck, that is a long time.
Not doing so well either at the moment which is why I popped by. Half the stuff that tams out I expect to do so because of science but the half I really like to get working is not doing great except some success with subtrops. I think the soil on my property is heavily infested with fusarium which seems to cause me problems during a certain time in the year when it's dryer and windy. A few things are pointing at this including some plants in the garden.
dude this is wild as fuck if it’s the soil. what a bullshit problem to be running into lmao
Quote:
rumfor69 said: Only thing I can think of is the technique when doing g2g in an sab. There's a lot more hovering over the receiver jar while the cement mixer movement is happening. It's a lot different than the clean outreach movement of a scalpel or syringe. I had lots of mold issues with g2g in an sab back in the day and only a flowhood solved it.
he’s doing agar to grain, not grain to grain. g2g seems like it could easily turn into a shitshow in an SAB for sure
-------------------- you're not the first to set foot here, just another =================================== i love glass petris & you can too!! posts i constantly refer back to new to mushroom cultivation?? read this!! ===================================
  🅃 🄴 🄰 🄼 🄲 🄻 🄸 🄽 🄶 🅆 🅁 🄰 🄿
Edited by hazyhorse (12/10/23 02:03 AM)
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Smellyhobbit
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Registered: 04/01/22
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Re: Recognizing and dealing with contamination [Re: Way]
#28576872 - 12/10/23 11:01 AM (1 month, 17 days ago) |
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Quote:
Way said: Your sterile work is usually spot on. You're not a noob and you know what you're doing. A2A and A2LC working without failure shows your transfers are fine.
You are running multiple different cultures so it can't be a culture related issue.
That leaves grain prep and sterilization right?
Have you left a control jar running without inoculating to see if it succumbs as well? If it does, it is definitely the PC giving up the ghost or an issue with the grain prep. If it doesn't, you've started doing something different during A2G that is causing the issue I would assume.
I’ll prep some grain today and set aside a couple of control jars. Give them a vigorous shake every week.
No different procedures in A2G. If it’s grain prep, why is the LC working? And I had a round of great success first time I tried this new grain prep followed by total A2G failure in subsequent batches. I’d say 2 batches of grain performed amazingly then nothing after.
Do you think it could be a grain QUALITY issue? I mean 2 hours in the PC should be sufficient, and even then the LC shouldn’t work if it’s a quality or prep issue, right?
I’m about to burn the apartment down and rebuild it out of stainless steel and glass so it can be flame sterilized
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rumfor69
Bodhicitta Cultivator



Registered: 08/05/11
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Re: Recognizing and dealing with contamination [Re: hazyhorse] 1
#28577474 - 12/10/23 05:36 PM (1 month, 17 days ago) |
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Quote:
hazyhorse said: he’s doing agar to grain, not grain to grain. g2g seems like it could easily turn into a shitshow in an SAB for sure
Ohhh yeah I see that now. Dunno where my mind was lol I'll just go look at stuff in the corner now
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