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Offlinebongoman
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Mold on 95% of plates after an SAB transfer session. WTF??!!
    #26334872 - 11/21/19 03:14 AM (4 years, 2 months ago)

I’m trying to understand where I went wrong. I had a bunch of clean looking T2 plates and performed a number of transfers to clean agar plates (they’d been sitting for a few days prior and no growth at all on them).

This was done in an SAB and nearly all T3 plates are now contaminated with dark moldy blotches.

I’m assuming that there was something airborne in my box? The blotches are spaced all around the plates not just one location.

I rinsed out the SAB first and left it wet. I used gloves and a mask but didn’t shower first. I think I maybe didn’t spray the air inside the SAB as I usually do just out of forgetfulness. Maybe that’s the issue?


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OfflinemeaculpaUIO
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Re: Mold on 95% of plates after an SAB transfer session. WTF??!! [Re: bongoman]
    #26334929 - 11/21/19 04:10 AM (4 years, 2 months ago)

what did you transfer from? picture?


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Offlinecurious.psychonaut
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Re: Mold on 95% of plates after an SAB transfer session. WTF??!! [Re: bongoman]
    #26334963 - 11/21/19 04:37 AM (4 years, 2 months ago)

Pics would help (or at least be fun to look at). SABs with dry walls work, just maybe not as good as misted ones, so not spraying can't be a reason for near-100% contam rate. (Rinsing it and leaving it wet does sound a bit sketchy though.)

How many days did the clean agar plates sit for? How many did you do in total and how many got the mold? I have a household fungus that is very consistent in popping out on agar at day 5. I've read here about contams that take more than twice as long to become visible. But the number of blotches and overall contam rate make me think there was something very wrong either with the procedure or the supplies you used. Maybe the sleeve was damaged and the petris weren't sterile, harboring a contam that became visible just after you transferred (aided by the fresh air it got when you opened the petris)?


--------------------
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InvisibleSpunkyMonkey88
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Re: Mold on 95% of plates after an SAB transfer session. WTF??!! [Re: curious.psychonaut]
    #26335006 - 11/21/19 05:17 AM (4 years, 2 months ago)

Quote:

curious.psychonaut said:
Pics would help (or at least be fun to look at). SABs with dry walls work, just maybe not as good as misted ones, so not spraying can't be a reason for near-100% contam rate. (Rinsing it and leaving it wet does sound a bit sketchy though.)

How many days did the clean agar plates sit for? How many did you do in total and how many got the mold? I have a household fungus that is very consistent in popping out on agar at day 5. I've read here about contams that take more than twice as long to become visible. But the number of blotches and overall contam rate make me think there was something very wrong either with the procedure or the supplies you used. Maybe the sleeve was damaged and the petris weren't sterile, harboring a contam that became visible just after you transferred (aided by the fresh air it got when you opened the petris)?




Why would rinsing it and leaving ot wet be any different than misting the walls?


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InvisibleBph
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Re: Mold on 95% of plates after an SAB transfer session. WTF??!! [Re: SpunkyMonkey88]
    #26335021 - 11/21/19 05:26 AM (4 years, 2 months ago)

Very well could be that your plates were not clean. You pour your own or do no pours? If they were no pours could be bad filter. If you pour your own could be bad sterile technique.  Hell you could have had clean plates/good filters and still bad technique. Could be the plate you made transfers from was fucked and it just showed it's true colors now. But honestly its hard to say with the info we have. I suggest starting a grow log a document every step. Folks here are wicked smart and can tell you what to do step by step if you ask.


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Offlinecurious.psychonaut
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Re: Mold on 95% of plates after an SAB transfer session. WTF??!! [Re: Bph]
    #26335103 - 11/21/19 06:32 AM (4 years, 2 months ago)

Quote:

SpunkyMonkey88 said:
Why would rinsing it and leaving ot wet be any different than misting the walls?




My thinking is that rinsing something so large (often in a different room) is never very clean: you pick up a lot of household contams in the process and on the way. If you let the water sit, poorly ventilated as it would be in a SAB, it's just not very hygienic.

But even if used immediately, a wet SAB makes me uncomfy: too much water. What if some drips from the ceiling, etc? Misting adds just 10-20ml of water and it's evenly distributed in fine droplets that will sooner dry out than drip...


--------------------
My LAGM2020 grow log



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InvisibleSpunkyMonkey88
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Re: Mold on 95% of plates after an SAB transfer session. WTF??!! [Re: curious.psychonaut]
    #26335145 - 11/21/19 07:20 AM (4 years, 2 months ago)

Quote:

curious.psychonaut said:
Quote:

SpunkyMonkey88 said:
Why would rinsing it and leaving ot wet be any different than misting the walls?




My thinking is that rinsing something so large (often in a different room) is never very clean: you pick up a lot of household contams in the process and on the way. If you let the water sit, poorly ventilated as it would be in a SAB, it's just not very hygienic.

But even if used immediately, a wet SAB makes me uncomfy: too much water. What if some drips from the ceiling, etc? Misting adds just 10-20ml of water and it's evenly distributed in fine droplets that will sooner dry out than drip...




Ok I can see your logic there, but as long as it's not dripping onto your trays I would think you could use toilet water with no issues because it basically just holds the tams down to the sides..

I dont use this method any ways...I'm pretty anal about sterile conditions. So I dont like the idea of working in a SAB with some dirty ass wet walls.. so I always wash it out with antibactrial dish soap, rinse and dry with paper towel, then I spray it in and out with ISO and wipe it down, then I lay 2 layers of paper towel down on the table, and spray first with ISO, then with soap water, then I put the SAB ontop of that, then I wipe everything down with ISO and put it in through the holes, then I have a little spray bottle of ISO inside that I use to spray the walls and air inside of it, then I let it sit for an hr before I work in it....

Think that's over kill? My house smells like a fucking distillery when I'm done...I worry that the smell if alcohol carries onto the hallway sometimes..


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InvisibleSpunkyMonkey88
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Re: Mold on 95% of plates after an SAB transfer session. WTF??!! [Re: bongoman]
    #26335150 - 11/21/19 07:23 AM (4 years, 2 months ago)

Quote:

bongoman said:
I’m trying to understand where I went wrong. I had a bunch of clean looking T2 plates and performed a number of transfers to clean agar plates (they’d been sitting for a few days prior and no growth at all on them).

This was done in an SAB and nearly all T3 plates are now contaminated with dark moldy blotches.

I’m assuming that there was something airborne in my box? The blotches are spaced all around the plates not just one location.

I rinsed out the SAB first and left it wet. I used gloves and a mask but didn’t shower first. I think I maybe didn’t spray the air inside the SAB as I usually do just out of forgetfulness. Maybe that’s the issue?




Were your dissection tools sterilized properly?


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OfflinemeaculpaUIO
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Re: Mold on 95% of plates after an SAB transfer session. WTF??!! [Re: SpunkyMonkey88]
    #26335169 - 11/21/19 07:32 AM (4 years, 2 months ago)

One way to control group this, is to bring one or two of your agar into the SAB, but not interact with them in any way. This will help you isolate where the error might be.

All the basics need to be crossed out. Proper SAB, proper flame sterilization that is red hot, that your spawn actually was a clean source, that your agar was properly sterilized.

By bringing some control group agar into the equation, it'll help remedy this.

If they stay clean inside the SAB, unopened, you know it was the inoculation and transfer that caused this. If they pop the green inside the SAB, it points to other vectors than what you're doing in the SAB. It could mean that your dishes are not properly packed, getting contaminated inside the SAB, but not by being opened. And it could mean that the contam was just waiting.

Also, you write that the dishes were sitting a few days.
How many days? It can definitely happen that a contam lurks for a few days or even longer before rearing its ugly face.

Seen things grow for two weeks, and then bam, green.


Edited by meaculpaUIO (11/21/19 07:38 AM)


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Offlinecurious.psychonaut
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Re: Mold on 95% of plates after an SAB transfer session. WTF??!! [Re: SpunkyMonkey88]
    #26335225 - 11/21/19 08:02 AM (4 years, 2 months ago)

Quote:

SpunkyMonkey88 said:
Why would rinsing it and leaving ot wet be any different than misting the walls?




Quote:

SpunkyMonkey88 said:
Quote:

curious.psychonaut said:
Quote:

SpunkyMonkey88 said:
Why would rinsing it and leaving ot wet be any different than misting the walls?




My thinking is that rinsing something so large (often in a different room) is never very clean: you pick up a lot of household contams in the process and on the way. If you let the water sit, poorly ventilated as it would be in a SAB, it's just not very hygienic.

But even if used immediately, a wet SAB makes me uncomfy: too much water. What if some drips from the ceiling, etc? Misting adds just 10-20ml of water and it's evenly distributed in fine droplets that will sooner dry out than drip...




Ok I can see your logic there, but as long as it's not dripping onto your trays I would think you could use toilet water with no issues because it basically just holds the tams down to the sides..

I dont use this method any ways...I'm pretty anal about sterile conditions. So I dont like the idea of working in a SAB with some dirty ass wet walls.. so I always wash it out with antibactrial dish soap, rinse and dry with paper towel, then I spray it in and out with ISO and wipe it down, then I lay 2 layers of paper towel down on the table, and spray first with ISO, then with soap water, then I put the SAB ontop of that, then I wipe everything down with ISO and put it in through the holes, then I have a little spray bottle of ISO inside that I use to spray the walls and air inside of it, then I let it sit for an hr before I work in it....

Think that's over kill? My house smells like a fucking distillery when I'm done...I worry that the smell if alcohol carries onto the hallway sometimes..




Not only overkill but some steps seem counterproductive, not to mention the impact on your health (inhaling so much iso adds up). For example, why spray iso on top of soap water coverage? To make it evaporate faster? That's not what you want. To sanitize it? But it's there to pick up contams! Also, keeping an iso bottle and iso-soaked paper towels in the SAB... you live dangerously, man! I hope you never introduce flame in that box.


--------------------
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InvisibleSpunkyMonkey88
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Re: Mold on 95% of plates after an SAB transfer session. WTF??!! [Re: curious.psychonaut]
    #26335254 - 11/21/19 08:11 AM (4 years, 2 months ago)

Quote:

curious.psychonaut said:
Quote:

SpunkyMonkey88 said:
Why would rinsing it and leaving ot wet be any different than misting the walls?




Quote:

SpunkyMonkey88 said:
Quote:

curious.psychonaut said:
Quote:

SpunkyMonkey88 said:
Why would rinsing it and leaving ot wet be any different than misting the walls?




My thinking is that rinsing something so large (often in a different room) is never very clean: you pick up a lot of household contams in the process and on the way. If you let the water sit, poorly ventilated as it would be in a SAB, it's just not very hygienic.

But even if used immediately, a wet SAB makes me uncomfy: too much water. What if some drips from the ceiling, etc? Misting adds just 10-20ml of water and it's evenly distributed in fine droplets that will sooner dry out than drip...




Ok I can see your logic there, but as long as it's not dripping onto your trays I would think you could use toilet water with no issues because it basically just holds the tams down to the sides..

I dont use this method any ways...I'm pretty anal about sterile conditions. So I dont like the idea of working in a SAB with some dirty ass wet walls.. so I always wash it out with antibactrial dish soap, rinse and dry with paper towel, then I spray it in and out with ISO and wipe it down, then I lay 2 layers of paper towel down on the table, and spray first with ISO, then with soap water, then I put the SAB ontop of that, then I wipe everything down with ISO and put it in through the holes, then I have a little spray bottle of ISO inside that I use to spray the walls and air inside of it, then I let it sit for an hr before I work in it....

Think that's over kill? My house smells like a fucking distillery when I'm done...I worry that the smell if alcohol carries onto the hallway sometimes..




Not only overkill but some steps seem counterproductive, not to mention the impact on your health (inhaling so much iso adds up). For example, why spray iso on top of soap water coverage? To make it evaporate faster? That's not what you want. To sanitize it? But it's there to pick up contams! Also, keeping an iso bottle and iso-soaked paper towels in the SAB... you live dangerously, man! I hope you never introduce flame in that box.




The ISO +soap water is for the opposite reason actually.. thw soapwater is for holding down the tams and reducing evaporation and the ISO is to minimize the amount of said contams.

I've tested the flammability of 70%ISO and it's really not that combustible you can spray it directly at a small flame and it will extinguish it...not sure about how that works with evaporation inside of a closed vessel though...hopefully it's ok 😬 but ya I never bring a flame inside...

Well let's hear your technique step by step, I really do wanna step away from using all that ISO because like you said the fumes are nasty! But man am I paranoid about the tams... between you and me I spent 6 months at an in house treatment center for people with OCD, and was hospitalized because I basically burned my lungs with bleach... I'm a recovering germaphob... so it's a deep rooted issue to say the least lol..


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Offlinecurious.psychonaut
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Re: Mold on 95% of plates after an SAB transfer session. WTF??!! [Re: SpunkyMonkey88]
    #26335276 - 11/21/19 08:19 AM (4 years, 2 months ago)

Quote:

SpunkyMonkey88 said:
I've tested the flammability of 70%ISO and it's really not that combustible you can spray it directly at a small flame and it will extinguish it...not sure about how that works with evaporation inside of a closed vessel though...hopefully it's ok 😬 but ya I never bring a flame inside...





No, it's not OK, the ISO in the solution evaporates faster than the water into extremely flammable fumes, turning the SAB into a bomb. If you use the search function, you'll see the report of one shroomer who visited the hospital because of it.

Quote:


Well let's hear your technique step by step, I really do wanna step away from using all that ISO because like you said the fumes are nasty!




Experienced growers who have done this for years should be a better source of step-by-step instructions. If you use the search function, you'll find a couple of SAB/sterile tech TEKs, I'd read at least a few of them.

---

Edit: some fun threads to read on the above topic. Also note that a SAB can be ignited from outside the box (iso fumes carry the flame).

https://www.shroomery.org/forums/showflat.php/Number/22586071/fpart/1/vc/1
https://www.shroomery.org/forums/showflat.php/Number/17947122/fpart/1/vc/1


Edited by curious.psychonaut (11/21/19 08:33 AM)


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OfflineFailboat
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Re: Mold on 95% of plates after an SAB transfer session. WTF??!! [Re: curious.psychonaut]
    #26335322 - 11/21/19 08:42 AM (4 years, 2 months ago)

How long dod you let everything sit before working? The SAB is all about STILL AIR and eo if you rush in to work it can be germy.


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OfflineSFS96
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Re: Mold on 95% of plates after an SAB transfer session. WTF??!! [Re: Failboat]
    #26335739 - 11/21/19 11:07 AM (4 years, 2 months ago)

:whathesaid: I used to get started too quickly and I’d have much more contamination then letting things settle for a bit


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InvisibleZifozonke
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Re: Mold on 95% of plates after an SAB transfer session. WTF??!! [Re: SFS96]
    #26335814 - 11/21/19 12:04 PM (4 years, 2 months ago)

I started experiencing the same problem in my SAB work
Went from clean transfers almost all the time-to batch after batch-full blown multiple contaminations of almost imaginable mould and bacteria.
I changed absolutely nothing in my process but was being savaged...
So I read and tried everything I could find on the contam forum.
Break everything down .
Bleach clean
Wet
Dry
Air spray ect...
Absolutely nothing helped.
Even tried moving my work to another smaller room ....
Same problems...
Then I thought I found my problem in my original room-a tiny bit of leftover LC in a jar that had a bit of mould on top.
So chukked it out -cleaned -sprayed ect ...tried again...same still...
Now my my dishes-They stay clean with no problem-for weeks-until opened.Then hell and brimstone arrives in the form of mould(s)

NOW THE ONLY THING THAT I CAN THINK OF THAT CHANGED IN MY SITUATION WAS THE SEASON....our dryish winters end-when spring-with high heat and rain with heavy humidity arrives....leaving the surrounding air thick with spores of moulds and bacteria ??(my theory??)...eish....makes me think it was not me-or my technique-but whats in the air??I dont know??

ANY CHANCE YOUR AREA HAS JUST HAD A SIMILAR SEASONAL CHANGE??or is my situation totally different??

Well I took the plunge and am now a day or two away from finishing up my hood Im building....hopefully mouldy dishes and working in a cramped SAB will be a thing of past soon....


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Offlinesporecap
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Re: Mold on 95% of plates after an SAB transfer session. WTF??!! [Re: SFS96]
    #26335821 - 11/21/19 12:07 PM (4 years, 2 months ago)

Reaching with your hands over an open agar plate also is a big risk, I think that's one of my main weaknesses right now where I need to be more careful:grin:


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Offlineverum subsequentis
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Re: Mold on 95% of plates after an SAB transfer session. WTF??!! [Re: sporecap]
    #26335836 - 11/21/19 12:15 PM (4 years, 2 months ago)

Upload some pics of your donor plates and the receiving plates. Did any of the new plates, that haven't been nocced up yet, show any contams?


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Invisiblealaskappalachian
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Re: Mold on 95% of plates after an SAB transfer session. WTF??!! [Re: SFS96]
    #26335937 - 11/21/19 01:07 PM (4 years, 2 months ago)

Quote:

SFS96 said:
:whathesaid: I used to get started too quickly and I’d have much more contamination then letting things settle for a bit




That is incredibly important IMO.  I can attribute early failures with agar to this.  I started pre-sterilizing everything and loading my box, then walking away for a while.. and voila: problem solved.  In reference to Zifozonke: I live in an unfinished cabin I've been building that has obvious black mold in parts of it where water damage has occured over time, and when conditions are right, I know I'm going to suffer from a higher spore load.  Creating an enclosed area with double-walled painter's plastc solved that problem.  I firmly believe people in older homes have problems with blooms when conditions are right that render their control mechanisms less-viable.  Just my 2 cents; but letting the air settle is key IMO.


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Offlinebongoman
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Re: Mold on 95% of plates after an SAB transfer session. WTF??!! [Re: verum subsequentis]
    #26336048 - 11/21/19 02:12 PM (4 years, 2 months ago)

Quote:

verum subsequentis said:
Did any of the new plates, that haven't been nocced up yet, show any contams?




Bingo. You'd think I would have checked that already but for some reason I'd just assumed that my new plates were clean as I've never had problems with my agar process previously.

9 out of 10 plates that were made at the same time but not inoculated are showing the mold.

So it's my plate making process that is suspect. I do no pour plates and can only think I didn't sterilise long enough as they were never opened after sterilising.


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Offlineverum subsequentis
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Re: Mold on 95% of plates after an SAB transfer session. WTF??!! [Re: bongoman]
    #26336088 - 11/21/19 02:28 PM (4 years, 2 months ago)

It's a bingo!


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