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InvisibleDizzy88
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Registered: 05/11/21
Posts: 82
Contaminated shoebox troubleshooting
    #27684715 - 03/06/22 07:23 AM (2 years, 10 months ago)

Hello fellow shroomers!

It's been a while since we've posted on the forum, because we were experimenting with our own ideas and all the knowledge we gained from reading the forum. We've had a few results since then, mostly succesful gourmet mushroom projects, but we've ran into some trouble with our cubensis project. Info on what kind of setup we are running here: https://www.shroomery.org/forums/showflat.php/Number/27395076

The problem at hand is with our cubensis grow in shoeboxes. We've tried to create a mycelium block from straw substrate in a shoebox twice now and both contaminated. The photos included are from the most recent attempt. The reason we are posting is because we'd like some external feedback on what went wrong. We have a lot of ideas but chances are yours might be better :wink:

Backstory: we bought a grow kit last year, golden teacher cubensis, and managed to get 1,5 flush before it sadly died (probably from cold temps). We extracted a clone and some spore prints from the first harvest fruits to process further. Made culture tubes, petri dishes, grain spawn in jars, etc. to experiment further. The agar to grain spawn part is going great, although it takes our jars about 3 weeks to fully colonize on room temperature.

Then we tried our first shoebox: 80/20% straw to horse poo. Both ingredients are in pellet form but they result in fluffy substrate when heat pasteurized. The shoebox did seem to colonize, but very slowly. Eventually, contamination started to appear on the surface and expanded. We used about half quart jars with spawn, about 4 of them. So our thinking was maybe our spawn to sub ratio was too low. We made more jars.

Second shoebox (on photos): we tried again with 8 half quart jars of spawn and 100% straw (pellet) substrate this time. Again, it took very long before we saw any mycelium appearing (we couldn't see inside because of condensation and we didn't open the lid). We waited until the block turned completely white and then opened: it wasn't the kind of mycelium we wanted to see :frown:

A few points:

- substrate 100% straw pellets at 55% hydration
- pasteurized in a closed bucket (straw hydrated at 55% beforehand) inside a large pan on 70 celsius (about 158f)
- layered straw substrate, grain spawn layer, straw layer etc. Covered up the grains on surface
- used a black garbage bag as liner
- taped the lid shut on the box with micropore tape to be clinical about it, lol

On hygiene: we use a SAB for any agar and grain spawn work. We try to be as clean as possible, flame sterilize, clean with alcohol etc. We've never had a jar of grain spawn contaminate this way. Our oyster bag projects almost never contaminate either.
For this shoebox project we used a bleach cleaned box, alcohol cleaned spoons, sniffed every spawn jar etc. We think slow colonization is our main problem, not hygiene.

The results: when we opened we saw a full block of contamination, we broke up the block and saw what is shown on the photos.
The mycelium started to grow from the grains, but it looks like it doesn't want to eat the straw. We had a separate bucket with leftover substrate which we tried to colonize as well, in that one the grains even fruited. But the mycelium in that one also didn't eat the straw. On the photos you can clearly see the layers of grain and straw we made and that the mycelium doesn't colonize beyond the grain. In the bucket photo you can see the mycelium growing upwards to find food and/or air to colonize or fruit. So the mycelium is growing but it's just not eating the straw.

Possible problems/solutions we thought of ourselves:

- substrate too wet > lower hydration?
- substrate too dense > maybe add vermiculite to increase air pockets?
- our cubensis strain just doesn't like straw > try coco coir substrate or something else (the kit we extracted the culture from was some kind of grain with rice flour and vermiculite)

- incorrect pasteurization? We did 90 minutes on 158f or 70c. > maybe try shorter pasteurization or lower temp? We also have the possibility to pasteurize with calcium hydroxide as we do with our oysters.

- degenerated culture? > we have two new strains cultivated from the spores we got off the first fruits of the kit. New genes reset senescence, right? We are growing the new strains on grain as we speak.

- we also contemplated making a tyvek/micropore filter on our shoebox, but from what we understand the block doesn't need air exchange while colonizing? When introducing fruiting conditions we'll just put another box on top of it and introduce air that way.
Another possibility is that we don't have a lot of space above the substrate when it's in the shoebox. The substrate depth is about 10 cm or 4 inches. The leftover room between the substrate and the lid is about 4 cm, almost 2 inches.



So to summarize... we plan on experimenting with smaller boxes now, because the shoebox failed twice. We feel like we need to experiment with hydration, substrate type and if nothing works ultimately try to continue with the new 'strain' we cultivated from the spores.

If you have any feedback for us, it would be very much appreciated!
I hope the post is coherent enough and thank you in advance for any tips/support.

Dizzy


Addition: found some pictures of the grain spawn we use. It's yellow oats at 60% hydration.
If any other information is needed we are happy to provide it, including pictures.


Edited by Dizzy88 (03/06/22 07:43 AM)

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Offlineschpat
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Re: Contaminated shoebox troubleshooting [Re: Dizzy88]
    #27685928 - 03/07/22 06:47 AM (2 years, 10 months ago)

No one has answered so I'll give it a shot
Quote:

Dizzy88 said:
We waited until the block turned completely white and then opened: it wasn't the kind of mycelium we wanted to see :frown:

For this shoebox project we used a bleach cleaned box, alcohol cleaned spoons, sniffed every spawn jar etc. We think slow colonization is our main problem, not hygiene.

The results: when we opened we saw a full block of contamination, we broke up the block and saw what is shown on the photos.
The mycelium started to grow from the grains, but it looks like it doesn't want to eat the straw.




You are correct that what has grown has grown from the grains, and also that it's not cubensis myc.  You've got trich  and it likely got hold of your grains because they were bacterial.

Quote:

Dizzy88 said:







I would say these jars are contaminated.  The first has a lot of uncolonised grains and the second has creamy myc with wet grains pressed against the glass.  I can't see your filters from the pictures so that may be a problem.

Also, as a beginner, mixing that many jars into a shoebox undoes the risk mitigation that using a shoebox is for.  If one of your jars is contaminated you contaminate the whole box.

My advice is to simplify
- carry on with the agar work, great choice
- stick with CV or plain coir these do not need pasturisation
- Spawn one myco quart per shoebox mixed with one quart of substrate and then add a 1/2 quart plain substrate layer.  Search for Shaper Dreamings SFF Shoebox Tek.
-leave your shoeboxes unmoddified.

Goodluck, you seem to be killing it on the gourmets.


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InvisibleDizzy88
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Registered: 05/11/21
Posts: 82
Re: Contaminated shoebox troubleshooting [Re: schpat]
    #27686252 - 03/07/22 01:16 PM (2 years, 10 months ago)

Thanks for the reply! Appreciate the tips.

Your first point: we recognized it's not the good mycelium immediately :wink:

The green mold is mostly on the surface though.

Our conclusion is the mycelium surrounding the grains is all cubensis myc, because we saw it grow in the jars from the agar wedges and like on the picture it started fruiting inside the jar as well as in the bucket when it reached the surface. Second jar picture shows this and the last bucket picture. That's definitely cubensis fruits.

Also there's a misunderstanding, which is my fault, on the first jar picture. We didn't use the jar in that condition, this is a mid-colonization picture (sorry :grin:). And it fully colonized with good looking myc after that picture was taken.

We let all the jars fully colonize while we were watching and smelled every one of them before use. So that's how we came to that conclusion. All our jars were already consolidating so that might explain the wet looking myc.



Nevertheless! We are prepared to keep jar contamination in mind so thanks for the advice.
We might try and tune down the hydration of the grain, right now we soak and drain them before sterilizing but maybe they're too wet.

Below is a picture where you can more or less see our jar filters. It's a tyvek filter with high temp sealant above a hole which is about.. a bit more than 1/8 inches? 4 millimeter.



Your point on the many jars in the shoebox is well taken. We decided on using a lot of spawn because we had high confidence in the quality of our spawn and because our first project as well as this one seemed to have failed because of slow colonization of the substrate.
That's why we are mostly looking for explanations why our myc doesn't eat the straw.

We thought more spawn = quicker colonization. So it's a trade off.
For next experiments we are planning on using more and smaller boxes like you advise, so that will mitigate this risk.



For the other points, we will look into the tek you recommend (link for reference).
and we'll try to keep it simple indeed. We have to eliminate the possibility that we have a genetically weak culture somehow.
Also added a picture of the culture we used on agar for those interested.

Trying out the coco coir and coco coir-vermiculite combo might reveal that the myc just doesn't like the straw, which would be good to know.

So anyway, thanks for the feedback man, really appreciate it!
We'll experiment with all these options and report back.

And if anyone has some knowledge to drop on the myc not eating the straw problem, we're open to it.



Dizzy

Edited by Dizzy88 (03/07/22 01:21 PM)

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Offlineschpat
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Re: Contaminated shoebox troubleshooting [Re: Dizzy88]
    #27687064 - 03/08/22 06:38 AM (2 years, 10 months ago)

Quote:

Dizzy88 said:
Thanks for the reply! Appreciate the tips.

Your first point: we recognized it's not the good mycelium immediately :wink:

The green mold is mostly on the surface though.





The green is the sporulating part of the mold.  The rest of the mold myc is white.  You pretty much only see the green on the surface where the spores can spread.

Quote:

Dizzy88 said:
Our conclusion is the mycelium surrounding the grains is all cubensis myc, because we saw it grow in the jars from the agar wedges and like on the picture it started fruiting inside the jar as well as in the bucket when it reached the surface. Second jar picture shows this and the last bucket picture. That's definitely cubensis fruits.




The pins and knots you have in that jar are early fruiting, this can be a sign of contamination too.

Quote:

Dizzy88 said:

Also there's a misunderstanding, which is my fault, on the first jar picture. We didn't use the jar in that condition, this is a mid-colonization picture (sorry :grin:). And it fully colonized with good looking myc after that picture was taken.

We let all the jars fully colonize while we were watching and smelled every one of them before use. So that's how we came to that conclusion. All our jars were already consolidating so that might explain the wet looking myc.





Smell is not the only indicator of contamination.  Here is a great link for spotting contams

Quote:

Dizzy88 said:
Nevertheless! We are prepared to keep jar contamination in mind so thanks for the advice.
We might try and tune down the hydration of the grain, right now we soak and drain them before sterilizing but maybe they're too wet.





What grain are you using and what is your prep method.  It looks to me like wheat, but I'm no grain expert. The large grains need to be boiled not just soaked.

Quote:

Dizzy88 said:
Below is a picture where you can more or less see our jar filters. It's a tyvek filter with high temp sealant above a hole which is about.. a bit more than 1/8 inches? 4 millimeter.



Your point on the many jars in the shoebox is well taken. We decided on using a lot of spawn because we had high confidence in the quality of our spawn and because our first project as well as this one seemed to have failed because of slow colonization of the substrate.




Again slow colonistaion could also be because of contamination.

Quote:

Dizzy88 said:
That's why we are mostly looking for explanations why our myc doesn't eat the straw.

We thought more spawn = quicker colonization. So it's a trade off.
For next experiments we are planning on using more and smaller boxes like you advise, so that will mitigate this risk.




Yeah perfect, that's what the small boxes are for.  You can also use Fhatlocks, they are very convienient for small batches.

Quote:

Dizzy88 said:

For the other points, we will look into the tek you recommend (link for reference).
and we'll try to keep it simple indeed. We have to eliminate the possibility that we have a genetically weak culture somehow.
Also added a picture of the culture we used on agar for those interested.

Trying out the coco coir and coco coir-vermiculite combo might reveal that the myc just doesn't like the straw, which would be good to know.

So anyway, thanks for the feedback man, really appreciate it!
We'll experiment with all these options and report back.

And if anyone has some knowledge to drop on the myc not eating the straw problem, we're open to it.



Dizzy




It's a pleasure to help, any time.


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InvisibleDizzy88
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Registered: 05/11/21
Posts: 82
Re: Contaminated shoebox troubleshooting [Re: schpat]
    #27687403 - 03/08/22 11:49 AM (2 years, 10 months ago)

Quote:

It's a pleasure to help, any time.



Thanks again for a very useful post!
The links to the contamination and Fhatlocks threads will be handy.


Quote:

The green is the sporulating part of the mold.  The rest of the mold myc is white.  You pretty much only see the green on the surface where the spores can spread.



Yes we are aware. The mold mycelium didn't reach far down however, so that's why we thought it was mostly a surface thing. In our observation it wasn't coming from the grains but it fell on top and spread from there. We're not 100% sure though.

The main thing is that our straw didn't colonize at all, the first pictures we posted show the shoebox at least 6 weeks after 'conception' and the mold mycelium grew out in only one week. Any thoughts on how fast the mold would have spread if it was in the grains? Maybe that would clarify, but it doesn't matter much anyway because we're over it and ready to experiment more :wink:


Quote:

The pins and knots you have in that jar are early fruiting, this can be a sign of contamination too.
Smell is not the only indicator of contamination.  Here is a great link for spotting contams




Thanks for the link! Maybe we were a bit blind to possible problems with the spawn because we've done over 100 jars and never had real problems with our gourmet bags we grew out of them. The only early pinning we have seen was on fully colonized jars and we don't see contaminated grow bags often. Almost never.

But maybe we were just unlucky with a few jars in the cubensis project, so we will use that thread to look more closily to how our jars are doing and try to pick out the bad apples.
We'll post some pictures of them here when we get there.


Quote:

What grain are you using and what is your prep method.  It looks to me like wheat, but I'm no grain expert. The large grains need to be boiled not just soaked.



It's yellow oats. We've used both black and yellow oats for a year and had similar results. At first we did soak & boil but we ditched the boiling when we didn't see any performance difference between the two methods. Now we only soak overnight and then sterilize. So we might go back to boiling and see if it makes a difference.

For clarity: do you advise boiling before or after soaking? We were doing soaking overnight and then boil (for about 20mins I think) before loading up the jars into the pressure cooker. I think we got that info from Youtube and thought the boiling only mattered to hydrate the grains a little bit better.


Quote:

Yeah perfect, that's what the small boxes are for.  You can also use Fhatlocks, they are very convienient for small batches.



This is a very nice tek. We have all those supplies so we will definitely be trying that!
The ziplocks are easy to handle, we use them for the gourmets as well.

For the different batches we'll try:

- straw pellets heat pasturized
- straw pellets pasteurized with calcium hydroxide
- the two above but mixed with vermiculite for more air

- coco coir
- coco coir with vermiculite mixed for air

And all of the above but with different percentage of hydration to see if water content is the problem.
Seems like a lot, we know, but we have prepared a lot of jars so we are ready to go full science on this one :tongue2:
And in case they all succeed (staying positive here) we have a dehydrator so we'll just dry those babies :wink:

You said coco coir didn't need pasteurizing? Because I've seen people use boiling water to prep their coco coir.
We might try that and we might as well try the two above mentioned pasteurizing methods on the coco coir too.
And we'll keep a close eye on the jars to see if we see any signs of contamination as per your link.

We'll document the spawn jars and corresponding batches and post the results :thumbup:
Thanks again!

Dizzy

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InvisibleDizzy88
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Posts: 82
Re: Contaminated shoebox troubleshooting [Re: Dizzy88]
    #27761329 - 05/03/22 11:42 AM (2 years, 8 months ago)

So we decided to do an update after some thinking and experimenting.

We are very thankful you reminded us of observing our grain spawn and seeing if it was contaminated and causing the problem with going to bulk. We also took the advice to go to coco coir instead of straw and keep it simple.
Here's the report of what happened.

First, we did an experiment with our grain spawn (oats). We didn't think it was the cause of the contamination, but we decided to try and improve our grain spawn anyway. We just started growing early last year, so we thought it would be good to go through our whole process again and improve it where we can.
The experiment was drying it better after soaking it for 24 hrs. We watched a few videos where people dried their grains after soaking and decided to try it out. We made 14 jars; 7 jars of soak-only grains and 7 jars of soak + dried for half a day.
The drying was done by spreading them out on paper towels on a table in a room with the windows closed.
After that we put the grains in jars and sterilized them at 15 psi for 2 hours.

We inoculated 2 jars king oyster, 2 jars pulmonarius, 2 jars citrinopileatus. All on the same day and same method.
The results made us very happy, the jars with 'dried' grains performed MUCH better than the 'undried' grains.

The jars on day 12: Citrino, king oyster, pulmonarius from left to right (left side jars are 'dried' grains)


Jars on day 19: Citrino, king oyster, pulmonarius from left to right (right side jars are 'dried' grains, except for the pulmonarius which has it on the left side)


The pulmonarius and citrinopileatus with dried grains were fully colonized by day 19.
So as you can see, your feedback helped us to improve our grain spawn by drawing attention to it. Thanks!

For our cubensis experimentation we turned to coco coir, because we didn't think the straw was working. For our strain at least.
We used Damion's Coir Tek, but we did freestyle a bit. We needed to extract some water after 'pasteurizing' and cooling the substrate because it didn't pass the squeeze test. We did so by wringing it in a towel.
More than a liter (quart) of water was removed.
That wasn't a fun procedure at all, lol. Next time we'll prepare the substrate on the dry side.

We dropped the coco coir in bags and proceeded as we would normally do with our straw bags. We decided on bags vs a shoebox this time, because you can more easily observe the mycelium growth when using a bag. We made 4 bags out of 1 block of coco coir, because the blocks aren't really dividable before you soak them :') We also had 4 jars of cubensis grain spawn.. so we used 1 jar per bag to avoid cross contamination.

The results of this experiment are looking good so far. The bags are 7 days old now and doing a lot better than the straw.
On the first two pics you can see the front and bottom of the bag colonizing. The last picture is the coco coir bag vs the straw. The coco coir bag was inoculated 10 days later!



We think these bags and especially the comparison with the straw bag is showing that our strain of cubensis is reacting a lot better on the coco coir vs the straw. We keep in mind the possibility our straw is too wet for this species, but if coco coir works we'll use that. We might re-test the straw in the future.

Btw, the spawn used for the cubensis bags wasn't included in our 'dried' vs 'undried' grain experiment. They were all 'undried' and took very long to colonize (3-4 weeks), so we can still improve there. We have watched our cubensis strain on agar and it takes the mycelium 3-4 weeks to colonize a 100mm agar plate as well. So maybe it's just a slow strain.

Any feedback and especially any tips on fruiting these bags are very welcome! We have access to a fully automated Martha tent and multiple shoebox sized tubs.

Thanks for reading and have a good day!

Edited by Dizzy88 (05/03/22 01:10 PM)

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Offlinenormalperson
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Re: Contaminated shoebox troubleshooting [Re: Dizzy88]
    #27761347 - 05/03/22 12:03 PM (2 years, 8 months ago)

you soaked your oats and then put them in a PC? Don't do that, all large grains should be cooked until the center is translucent or they will not sterilize properly. Also, Oats suck almost as bad as corn, i suggest you try wheat or rye.

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InvisibleDizzy88
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Re: Contaminated shoebox troubleshooting [Re: normalperson]
    #27761394 - 05/03/22 12:43 PM (2 years, 8 months ago)

Quote:

normalperson said:
you soaked your oats and then put them in a PC? Don't do that, all large grains should be cooked until the center is translucent or they will not sterilize properly. Also, Oats suck almost as bad as corn, i suggest you try wheat or rye.




Thanks for your response.

We did exactly that, yes. We've seen many people do it online, on video and with results. We were mimicking them.
By cooking do you mean boiling on atmospheric pressure in a pot before sterilizing? We've seen people do that as well.
Does this replace soaking or do you advise both? We thought the soaking was to activate contaminants before sterilizing to kill them. What does the 'cooking' do?

And why do oats suck, if I may ask? We're open to using any kind of grain, but again.. many people seem to use oats to great effect.
What is your experience with them compared to other grains? What difference did you see?

Thanks

Edit: did some searching and found this TEK by bodhisatta. He doesn't soak but boils the grains for 30-45 mins before draining them for 30-60 minutes. Is that what you mean and do you recommend this or would you add some information?
https://www.shroomery.org/forums/showflat.php/Number/24126032

I also found this video by PhillyGoldenTeacher. He uses the same technique; boil, drain/dry and then sterilize.


We'll definitely try this out.

Thanks for pointing it out

Edited by Dizzy88 (05/04/22 01:31 AM)

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InvisibleDizzy88
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Registered: 05/11/21
Posts: 82
Re: Contaminated shoebox troubleshooting [Re: Dizzy88]
    #27762302 - 05/04/22 02:03 AM (2 years, 8 months ago)

Here is an alternative method to prepare oats by Mossy Creek Mushrooms. I think we were mimicking his procedure.


We were getting good results with almost no contamination on oyster mushrooms with this method, but we found out with this recent drying experiment that the results were better if you dry the grains or at least strain them a little bit better.

I’d like to see data and counterpoints on different methods and grains. But seeing we can find so many different ways to prepare oats (sometimes conflicting ones) we’ll have to try it out ourselves and see what works best for us.

Anyways, thanks for reminding us where to look to improve. There's so many aspects of the whole cultivation procedure that you sometimes forget one while looking at another. Feedback is invaluable for that.

:smile::thumbup:

Edited by Dizzy88 (05/04/22 06:53 AM)

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