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InvisibleWinter_Bohemian
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How to salvage Contaminated Mycelium Cakes and Jars?
    #27784862 - 05/19/22 07:57 PM (2 years, 7 months ago)

MY PROCESS


3/29/22
I used a Pressure Cooker to sterilize my jars and grain following a PC TEK
I Inoculated 12 jars following a PF Tek
I opened two Agar Plates as a control (one Agar Plate was injected with spores from one of the syringes the other was open air)
I used a sterilized grow tent with a LFH


I sprayed everything down with two diff. types of Lysol, Alcohol, Hydrogen Peroxide
I put the laminar flow hood in and let it run while I took a shower.
I put in only the items I was going to work with.

I then came out with a special cap on; Fearing my clothes had contams on them from them I stripped and put on some gloves which were sprayed with alcohol; I flame sterilized between every syringe, two holes on each side 1~1.5ml. I ran out of spores; came back in (repeated the same process before getting in). Sprayed alcohol and hydrogen peroxide everywhere each time. Then flamed between each one.

Opened the green plate for a few seconds and closed.

Took needle off agar plate and accidentally overshot.

When I was done I came in and I put polyfill on the doors; leaving the jars outside in the martha styled grow tent, lows will be 40F highs 60F, after that it will be warmer.


I was fearful that the jars would contam if I carried them back inside the house and wanted to be certain nothing was introduced so I left them in the tent and checked on them infrequently to avoid disturbing them.

4/11/22 - Day 14 no change
Decided to wait at least 3-4 weeks before giving up. No contams appear on the plate.

4/30/22 - Day 33, 4 weeks
Mycelium growth is seen in a few jars; mycelium has not spread through the whole jars yet though. No contams were visible but I did not turn the jars around. Some jars still show no growth.

5/9/22 - 42 Days, 5 weeks
More mycelium is apparent; nights have been chilly. Mild during day. No extreme temps noticed.

5/19/22 - 52 Days , 6  Weeks, All Contam

1. Some Jars Never contamed but also never showed mycelium growth.
2. Some jars were HALF Mycelium HALF Trich. Contam
3. Some Jars had some Contam, with some growth mixed in it (like the contam I got before using LFH inside tent)
4. Agar Plates never contamed, but nothing grew on the plate that DID have mycelium spores added to it
5. Contam was 'black wet' spot on patch of white mycelium **(or)** Green Trichoderma
6. Jars that show no contam only have SOME Mycelium growth, might have contam that I can't see
7. All Jars removed from the container they were kept in showed white mycelium on one side, contam on opposite

Spore syringes had been kept in the fridge for at least a year.
Same spores in different syringes from the same vendor.

MY QUESTIONS
I don't have anymore spores and can't purchase anymore at this time.

1. Some of the mycelium looks like strong white mycelium... Are any of those cakes salvageable?
2. Can I grow them outdoors?
3. Where did I go wrong? Was it the outdoor temps? Did it take too long to colonize due to those temps? Did I contam. them when I came to check on them?


Thoughts (while I try to troubleshoot):

I didn't think anything was wrong with how I pressure cooked them because some jars never got contam and none of the plates contamed. I don't think that I contam. them because the plates I had opened and worked with never got contam, and I followed the same process for all of them. I don't think my LFH has a hole on it and isn't working, because I had a control plate where all I did was open and shut it to see if the LFH was defective or not (it was not). I also had it inspected and it was working fine. I thought it could be the spores themselves, but the agar plate that I injected with spores never contamed. So maybe water was sterile but the spores were dead?

This is the closest I've gotten to actually getting almost contam. free process. I got so close. Not sure what to do next. As of this moment I removed the contam jars from the same tub as the cakes; but still inside the tent.

I would like to not throw the jars away and hope to place them into one of my composts or on some cardboard in one of my piles and then wash and swap my clothes once indoors, come out and lightly water. My compost is made up of straw, various animal manure (chicken, ducks, horses, cows, vermicompost, frass) and cardboard. I also have compost piles that are all cardboard. Hoping they can be salvageable even if I throw them into a pile outdoors and see what they become.

Any help would be greatly appreciated!


PHOTOS:


ITEM 1 - Front/Back






ITEM 2 - FRONT/BACK







ITEM 1 and 2 together -



3 Jars I think made it? (slow growth, rotated around)












NOTE: The blue agar has pieces of plastic shavings or cotton that fell from the holes (I used cotton filters on the holes of a SAB where I kept the jars inside within the tent in hopes of limiting contam :undecided: ).

Total: 5 Jars out of 12 appear to be even semi-viable (no photos of remaining 7)

Edited by Winter_Bohemian (05/20/22 08:06 AM)

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Offlinehazyhorse
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Re: Is it Salvageable? [Re: Winter_Bohemian] * 2
    #27784890 - 05/19/22 08:34 PM (2 years, 7 months ago)

ok that was a lot lol

one, you're going way overboard in all the wrong ways for trying to limit contamination. if you're using a true LFH you should be fine. i work in my SAB without showering & don't even spray it with isopropyl or anything anymore. i know a LFH is different but the same concepts should apply. it's important to be mindful & use good technique, but you're not doing surgery. also, lysol & hydrogen peroxide aren't needed nor particularly helpful. 70% isopropyl is all you need for anything. i don't even use gloves in my SAB, just don't hover hands over stuff & don't create eddies in the laminar flow

you can pick up & transport/move your jars around. i'd be careful with them, but you're not going to contaminate them from just moving them from one place to another. just don't shake or rough them up in transport & you should be fine. once they're in the jar & it's properly sealed you're good to go. you could move them in a tote or something if you're really worried about the outside air

those first two "items" look contaminated with bacteria to me, can't really see the rest of the pictures well enough to tell anything about them. if your agar plates didn't contaminate then your sterile process there was fine, those temps are really low though & germination was probably slow/non existent because of that. if you take the agar plate with spores on it inside to a warmer location, maybe something will spring to life. it looks like you got some myc in the jars in some form, so the spores must have germinated in some capacity. the temperature was probably a huge factor, 60-80f is ideal. something was off if that many PF jars contaminated though. how long did you PC for? did you vent the cooker before bringing it to pressure?

take clearer pictures of the other jars & you might get some better answers on if they look good or not. if that plate doesn't germinate for awhile after you bring it to a warmer area, i can get you a spore print totally for free if getting another syringe is out of the question.


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Edited by hazyhorse (05/19/22 08:44 PM)

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Onlinenektar61S
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Re: Is it Salvageable? [Re: Winter_Bohemian] * 1
    #27784912 - 05/19/22 08:48 PM (2 years, 7 months ago)

"Lysol, Alcohol, Hydrogen Peroxide"

Lysol has bleach-like chloride chemicals. This might make toxic gas if it mixes.

Just use 70% ISO alcohol. That's what 99% of the people growing tons of fat shrooms here use.

Not 90%, use 70%. Weird, but 70 is better.

Don't try to save anything with green. I would even waste the jar and toss those without opening them. Green means mold spores, can contam the room.

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Offlinehazyhorse
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Re: Is it Salvageable? [Re: nektar61]
    #27784930 - 05/19/22 09:00 PM (2 years, 7 months ago)

you can just open the jar & dump it out in your kitchen or outside, then wash the jar with soap or water. no need to waste the jar. trich spores are everywhere, you don’t wanna leave a super moldy tub in your room for weeks but it’s not gonna ruin anything to open a jar


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InvisibleWinter_Bohemian
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Re: Is it Salvageable? [Re: hazyhorse]
    #27785365 - 05/20/22 08:05 AM (2 years, 7 months ago)

I apologize for the length of the post; I thought if I just showed a picture of my growth and did not share the process it took to get there that I could be helped in narrowing down how the contams got there. Most of the mycelium looks good. It's not until I turn the jar around that I see contams in a spot or on half the jar.

I appreciate the offer for a spore print. I suspect I would end up in the same situation as I am now with contams. Everything I've every tried to grow from spores leads to contams. This is the first time though I've ever gotten fully or half-colonized jars! So even as I am disappointed I feel I've made progress.

I felt I had to be "extra" regarding hygiene for this particular grow to rule out source of contam. If it contamed I would have worried that my hair or clothing had been a vector. I don't plan to be extra every time, I understand 70% alcohol was the only thing needed and likely will only use LFH and 70% alcohol next time as my agar plates got zero contams so the LFH is working! The Pressure Cooker I followed careful step by step instructions. Got it to 15 PSI and then let it cook for 60 minutes (I believe a bit over). Then I turned it off and let it sit for 8 hours overnight. Some jars have zero contams and zero growth so I'm doubtful that it's my grain -- why would some jars not contam while others did when inoculated in the same conditions?  (genuine question) My thought is different syringes = different results? (guess?)

As a layman I suspect:

  • Outdoor temps contributed to slow growth (will remove the jars with no contams, and plates inside now to see if I get more growth)
  • Slow growth of mycelium gave time for mold to grow (it'd been there for 6 weeks)
  • Same Spores: Different Syringes = Different Results (?)
  • The Mycelium looks good and pins will grow where the mycelium is good (if it is indeed good).
  • I can bury it in a cardboard/manure compost outside and see if it pins.
  • A bleach soak might help save the colonized mycelium but kill the bacteria. (could I do an indoor grow then?)


My primary question (now) is: "How can I salvage contam. mycelium?"


  • Can I soak the mycelium in 1:200 bleach:water solution for 10-30 minutes before soaking them in water for 24 hours? If I did this would it kill the spot I saw on the fully colonized jars; for the jar with Trich on one side and mycelium on the other could I do the same in an isolated containter; soaking only half of the myc that grew in a bleach solution?
  • If I buried them in my cardboard/manure/straw pile what maintenance would be required? (or) What outdoor Tek would be recommended to ensure success amidst temp. variations or watering requirements?
  • Could I transfer the good mycelium to an agar dish in front of an LFH hood? Can you open contam jar in front of a LFH inside a clean area? (or would a presoak in bleach be recommended before this attempt). (or would it be best to do an outdoor bleach soak before working in front of an LFH).
  • Even if the Mycelium was not safe to consume, in theory, couldn't I do Agar transfers or pin transfers until they were no longer toxic (Mycelium can clean contaminated soil - could toxic mushrooms not themselves overtime become clean)



The mycelium appears to colonize half or all of the jars. Some of that mycelium looks like really good mycelium. I would like to bury it (or) explore a bleach solution and am hoping for guidance from anyone with experience managing contams.

I guess the how it got that way doesn't matter to me so much as, OK, it's here, what steps to take to save and maintain the mycelium I do have? (without throwing it out), how to cultivate it. Does anyone on this board have experience growing from contam mycelium outdoors, cleaning it up via bleach to grow indoors, or doing transfers grain to agar transfers until all traces of contam are gone?

My understanding from RR's posts/replies and mushroom company videos is that in the wild, mold colonies grow side by side with Oysters, Shittake and other safe to eat mushrooms, and that no one has ever died from eating them. So how do I work with what I've got?

(looking for a TEK or instruction guide). Or would I largely be in the 'on my own, cross my fingers and hope' category for this one =).

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OfflineThe Tao
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Re: Is it Salvageable? [Re: Winter_Bohemian]
    #27785464 - 05/20/22 10:01 AM (2 years, 7 months ago)

Which PC tek did you follow? Which PF tek did you follow? Can you post a picture of the LFH? Where did you get the 1:200 bleach idea?
I make spore syringes and inoculate my BRF jars in a SAB on top of my washing machine. I spray my hands and SAB down with soap and water. The jars colonize in the basement with my plants.
You’re really over thinking this.

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InvisibleWinter_Bohemian
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Re: Is it Salvageable? [Re: The Tao]
    #27785615 - 05/20/22 12:00 PM (2 years, 7 months ago)

Quote:

The Tao said:
Which PC tek did you follow? Which PF tek did you follow? Can you post a picture of the LFH? Where did you get the 1:200 bleach idea?
I make spore syringes and inoculate my BRF jars in a SAB on top of my washing machine. I spray my hands and SAB down with soap and water. The jars colonize in the basement with my plants.
You’re really over thinking this.




Thanks for your reply; I understand that 70% Alcohol and LFH was all that was needed to be done; unfortunately after several years with a series of contams I needed to remove all doubt of where my contams were coming from. Being "Extra" is what I needed to do for my own sense of ease. Given time and confidence I will ease on it, would being extra precautious have caused the contam?

I used: Bod and Frank's Teks are the guides I used regarding Pressure Cooking and PF Tek's.

I use Bonsai LFH from Midwest.

Here is where I got the idea to use bleach from:

Bleach:
https://www.shroomery.org/forums/showflat.php/Number/611022/fpart/all

https://www.shroomery.org/forums/showflat.php/Number/2899593

https://www.shroomery.org/forums/showflat.php/Number/2176149#2176149



Mold Impact by RR:
https://www.shroomery.org/forums/showflat.php/Number/7666274#7666274



I have never had a contam free culture except when I did the culture in a lab. I have been trying to grow mycelium since 2018. Have made more attempts than I can count. That is with showering, soap, water, alcohol, gloves, no gloves. Error vectors were sometimes obvious, somtimes not.  Each time I write down the mistakes I've made or think I made and try again. Recently I took some in person classes at an in person mushroom company and got hands on experience from people who do it for a living both outdoors and indoors. 70% alcohol and an LFH with a flame is all that is needed. Still I took extra precautions because while extra, it doesn't hurt anything and gave me peace of mind.

This is the first I've ever seen so much success built on the backs of all my prior failures. So many speak about how easy it is to grow mushrooms and how they got it in a few tries (certainly less than several years!), dang, I feel very inadequate and illiterate. Like the dumbest fuck. I've done more than I can count. It has not been an easy journey from spore to mycelium to pins. I'm still trying. Each time I get a little bit closer but I'm at the end of my rope: Out of cash, out of spores, and a growing sense of pain to keep failing. What I have left are these beautiful white tendrils of hope filling half or most of my jars with these specks of disappointment (contam). I'd like to pursue it even if it ends in failure, but ideally with guidance from someone whose done something like this before >.<.

Even if consensus says to throw out my jars, there are a small group of people on shroomery who say they've had success with moldy mycelium, cutting away contams, RR himself says it won't kill you (paraphrase). Where I'm at is that at this point contam free mycelium would be nice but after several years; maybe I've got a moldy thumb and it's time to work with nature instead of against her.

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OfflineThe Tao
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Re: Is it Salvageable? [Re: Winter_Bohemian]
    #27785652 - 05/20/22 12:23 PM (2 years, 7 months ago)

Those threads are all 14+ years old. Keep to current posts.
If you give yourself more vectors then you give yourself more possibilities that you have to look at for potential contamination.
I don’t know what your jars look like now but you could possibly top fruit them and get a couple of mushrooms. That would allow you to make some prints and start fresh.
Simple it down. Doesn’t sound like all the extras are giving you any peace of mind.
Any time you need a print, I made some yesterday.

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OfflineTheTimelessDon
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Re: Is it Salvageable? [Re: The Tao]
    #27785694 - 05/20/22 12:52 PM (2 years, 7 months ago)

So much to read... But yes it's salvageable.  Tao is on the right track with the top fruits.  My first run was 6 jars, all 6 half-full of mold... I took 2 of them and birthed them, separating the mold off, and put 2 half cakes in fruiting chamber.  They did great, and I got clones, prints, etc...  Definitely worked!

2 Jars were birthed into a flower pot with some random bush in it.  Soaked it down and covered with a trash bag for humidity, while spraying if needed.  I got some fruits from that too!  After that first lil flush, I put the pot outside.  Now, I just found some HUGE shrooms, dead as fuck cause I didn't think I'd get a 2nd flowerpot flush.  Got to them old and rotten, but if I only would have seen it cause the dried and shriveled ones were massive. 

2 jars were just way more mold than myc, so I threw them away.  I know I could have saved a few mushrooms out of them.  But by then, I already learned that it wasn't worth my time.  Fresh, healthy jars/tubs already set up on the way, and not too worried about saving.

So to answer your question simply, yes!  Definitely yes!  Is it worth it??  Only you can tell us that based on what you wanna do!  Good luck!

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Offlinehazyhorse
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Re: Is it Salvageable? [Re: TheTimelessDon] * 1
    #27785751 - 05/20/22 01:21 PM (2 years, 7 months ago)

oh you’re fine on the post length dog! you did the right thing providing all that detail & i didn’t mean to make it seem like it was too much. it really helps in trying to figure out what’s going on so i do appreciate it, but a lot of people won’t thoroughly read through suuuper long posts either. but again having the detail is super nice



salvaging contaminated stuff isn’t necessarily something you want to do with this hobby. sometimes stuff like a tub breaking out with trich with a few days left on the flush is worth letting ride, but if you’re getting colored mold in jars it’s best to just start over. that being said, like others said you can def get mushrooms out of a lot of situations. it’s just usually not worth your time to try & save. you could bury them outside or top fruit the ones that are just bacterial like everyone else said. everyone else covered pretty much everything, the only thing i really wanted to add is if your agar plates stayed sterile you should have a good chance of getting spores from a print to germinate. your agar plates staying clean means you did something right, & i think a quality print from someone off here would serve you well on some agar plates. sorry you’ve been struggling so much with this hobby, i don’t know exactly where you went wrong with the jars & it’s hard to say since i did ok when i first started so i didn’t have much to trouble shoot, but if you keep at it i’m sure you can get something to work.


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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!!
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InvisibleMysticMycologist
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Re: Is it Salvageable? [Re: hazyhorse]
    #27786061 - 05/20/22 06:00 PM (2 years, 7 months ago)

“As long as you’re trying to save dirty grows, you won’t learn how to do clean grows.” - some OG on the boards.

Anexic spawn is king. You will be surprised with the shit you can get away with if your spawn is clean.


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InvisibleMTZ
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Re: Is it Salvageable? [Re: MysticMycologist]
    #27786186 - 05/20/22 08:37 PM (2 years, 7 months ago)

Truth. Also I'd like see see the flow hood. Could very well be just working in the open air.


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InvisibleWinter_Bohemian
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Re: How to salvage Contaminated Mycelium Cakes and Jars? [Re: Winter_Bohemian]
    #27786517 - 05/21/22 05:20 AM (2 years, 7 months ago)

Thank you for the replies and for the hope. I was feeling really down, but feeling much better now.  I wish I had checked the forums before I proceeded as there were great tips given (For instance I wouldn't have dunked them would have just sent them straight top fruit or buried somewhere instead of wasting so much time with them). My perspective has now changed from, "I just need something not to end in failure" to I'll treat it as a "start towards success" and part of the learning experience. I am much closer to my end goal now than I've been since when I started in 2018. First contam. free Agar plates! YAY!

I will take the advice and try to get my hands on spores / spore prints (I feel like I am so close to getting something contam. free that I'm doing my disservice to not focus on building up on that momentum). The contam. ones will end up buried / top fruited somewhere while I work on acquiring spore prints / spores. Admittedly I haven't read up on spore prints the same way I have with agar plates and syringes. If I acquired a spore print would I just scrape some of it into the agar? How much just a piece? I suppose I need to start looking up spore prints to agar Teks now!

When I do work again with the agar and spores, I'm going to turn the LFH on in it's enclosed space, and work with my materials and 70% alcohol nothing more. I feel like if I  had inoculated 12 Agar Plates instead of 12 PF Jars that I would have had more success than failure and would still have my spore syringes.

My question now is: Is the Birthday suit necessary if you have a working LFH and your clothes are freshly laundered but you have to walk outdoors to get to your work space?


@MTZ - I have a Bonsai Flowhood. I  had it inspected by a professional grower who turned it on and verified it worked. Then I did a test and opened two agar plates in front of the agar flow hood; one I inoculated the other I did. No contams.

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Offlinehazyhorse
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Re: How to salvage Contaminated Mycelium Cakes and Jars? [Re: Winter_Bohemian]
    #27786782 - 05/21/22 11:25 AM (2 years, 7 months ago)

glad you’re feeling inspired!! that’s a good way to look at it i think. when working from a spore print that’s essentially what you do, what i do is flame my scalpel, cool it off in the agar plate, then scrape it on the foil to get some spores on it, then i drag it back across the agar. the proper way to do it is to use an inoculation loop & there’s a certain way to streak it that’s considered best practice but getting spores on the plate is the main goal. also you def don’t need the birthday suit. i rarely ever shower beforehand & if i do it’s generally coincidental. just iso up your hands & arms & you should be good to go

like you figured, im pretty sure your LFH is fine considering the agar plates didn’t contam


--------------------
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!!
===================================

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InvisibleWinter_Bohemian
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Re: How to salvage Contaminated Mycelium Cakes and Jars? [Re: hazyhorse]
    #27792393 - 05/25/22 01:58 PM (2 years, 7 months ago)

Thanks Hazyhorse; I did indeed purchase some new spores. In addition to that I brought the sealed agar plates in to be at room temp. One had mycelium spores in it (the charcoal one); and the other was just open and shut in front of the laminar flow hood. My hands had been soaked in alcohol and allowed to dry before doing so and I was freshly showered.

The Agar Plate with charcoal that had inoculate in it did not mold, but there is also no mycelium growth.
The Agar Plate without charcoal didn't mold outdoors, but DID mold once brought inside.

There are 3 jars that I saw no signs of mold on with mycelium; the mycelium so far shows positive growth on at least one. I didn't see mold with the other two.

I'm not quite sure what happened or what went wrong with the cleanliness. The person who inspected my laminar flowhood said it worked fine. I felt I was meticulous with my cleanliness. That the agar plate with charcoal didn't mold doesn't mean there wasn't contamination in it due to the charcoal. Maybe given more time contaminates will show up in the charcoal agar plate as well.

Perhaps it is as MTZ Suggested "Could very well be just working in the open air."

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