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Invisiblewildlifesc
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I give up ;( :( :( ;( ;(
    #28457579 - 09/03/23 07:02 PM (4 months, 22 days ago)

I don't know what to say... I've been trying for over a year and have not been able to grow anything from spore.  Not even with PF tek, and not even see a pin.  I have just decided to trash all of my remaining GT cultures (as you can see in the pictures below all the agar dishes have the lid removed) because I no longer know if those are even mushroom mycelium.

Let me give a brief background - these are the things I HAVE been able to do with various levels of success:
1) Fruiting: starting with commercial spawn I have no trouble making bulk sub and fruiting edibles with decent yield.  So I think my fruiting conditions are okay.
2) I have been able to grow from a commercial LC.  Lion's Mane, LC to grain, then to bulk and fruit.  Yields weren't great but I got some fruits.
3) I have been able to clone an oyster one time.  Fruit biopsy to LC, to grain, to bulk.  The block took a while to fruit, I got tired of it and put it outside but eventually it gave one flush that wasn't bad.
4) I have been able to get a cloned Chestnut to pin one time.  Fruit to agar, to grain, then to bulk.  The block started pinning but then got trich and the fruits never matured.
5) I feel that my agar techniques are pretty good.  I use a SAB.  When I first started I got bacterial contam and green mold often, but after I made some improvements, now I pretty much NEVER get bacterial or green mold (I don't think I got any in the past 40 dishes I inoculated).


Now, having said those, I have not been able to grow ANYTHING from spore.  I've tried two types of oysters, a Lions Mane, and GT, many times.  I would go through the process (spore -> agar -> grain -> bulk) and everything would look fine, but the fruiting blocks would just sit in the fruiting chamber for weeks and do nothing, then eventually get contaminated.  Finally I decided to just do PF tek, since I felt pretty confident that my agar is good, then if I can get a fruiting body from the PF tek that I can clone, perhaps they will be more eager to fruit in a bulk fruiting block.  I tried 3 different mushrooms in 4 PF tek blocks, none of them fruited.

What ultimately pushed me to despair is this: I have some prepared agar that I keep in a ziplock bag for convenient use.  They are completely sterile so they keep for a long time (it could take me several months to use them all).  I check them from time to time for contam - occasionally I would get a dish that get bacterial spots.  Now, the last time I checked there was a dish that had something growing.  The disturbing thing is, if I didn't already know this was contam, I wouldn't be able to tell that it is not mycelim (see two pictures here).  I left this dish growing for several more weeks, it stayed looking like this and never turn green or fluffy or pink or any other clearly mold-looking thing.



This made me question everything that I had been doing.  Have I just been growing contam all along not being able to tell them apart from mycelium?  Please check my sanity here, these are my GT dishes, do they look like mycelium or just contam?  (Note: these have been sitting in the fridge for over 8 months so they could look a little weird for that reason)



These are my GT PF tek blocks, they have been sitting in a fruiting chamber for 2.5 months, do they look like mycelium or just contam?



What the hell am I doing wrong?  How do I distinguish between mycelium-looking contam and actual mycelium?


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Offlinephenyl
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Re: I give up ;( :( :( ;( ;( [Re: wildlifesc] * 1
    #28457590 - 09/03/23 07:18 PM (4 months, 22 days ago)

None of those dishes look right. Errors compound. It's very frustrating. Mold and bacteria suck.

If you're into it, dump everything, make more plates, verify they're clean after a few weeks of storage and your SAB keeps clean things reasonably clean with a plate exposure test thereafter, and go from there. Obviously make sure you're sterilising your agar properly too - I had issues with a batch that I forgot to PC (don't ask...) that made my cultures look nasty af.

If it makes you feel any better, golden teacher is mediocre anyway and I had a tonne of problems with deli cups contaminating after a while too and culturing white mold. I'll give you a hint: go for the nice national geographic quality ropey white rhizomorphic growth instead of whatever germinates until you can reliably spot healthy cubensis mycelium. One drop of spores on a plate streaked in a pattern so the thing doesn't get swamped in germination and become confluent and indistinguishable.


--------------------
The fool who persists in his folly will become wise.


Edited by phenyl (09/03/23 07:35 PM)


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OfflineKinoko314
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Re: I give up ;( :( :( ;( ;( [Re: phenyl]
    #28457660 - 09/03/23 08:18 PM (4 months, 22 days ago)

Yeah, the agar doesn't look good.  I see a couple small pins on the pf tek.  Are the small pins aborting, or stopped growing?  Maybe they'll still get bigger.

I don't know if it's the lighting, but the top of the cakes look kind of gray.  I'm not sure what would cause that exactly, but it doesn't look great.

What kind of fruiting chamber are you using for the cake?  Try a water tub. Also, how big is that cake?


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Invisiblewildlifesc
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Re: I give up ;( :( :( ;( ;( [Re: Kinoko314]
    #28457705 - 09/03/23 08:52 PM (4 months, 22 days ago)

I've never been able to get ropey white rhizomorphic growth on anything.  I wish I could, then I'd know it's mycelium for sure.  But I don't know how to make it happen. 

If my agar is contaminated, it's so weird that they only get the "mycelium-looking" contam and nothing else.  I never get bacterial blots nor any green/pink mold, and rarely the fluffy white mold, only stuff that looks similar to mycelium.

Maybe I'll start over with PE instead of GT and see if I have better luck.  I have some PE spores but have not tried growing those.

@Kinoko314 where do you see pins?  I only see vermiculite at the surface, some of them are half-grown-over by mycelium (or whatever is growing).  I just threw out the cakes an hour ago into the backyard.

The cakes are about 2.5" diameter and 3~4" height, so pretty small.  I use a simple mono-tub kind of chamber.


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OfflineKinoko314
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Re: I give up ;( :( :( ;( ;( [Re: wildlifesc]
    #28457738 - 09/03/23 09:21 PM (4 months, 22 days ago)

I swear I saw some. . .but I guess I'm crazy.

Maybe I'm still tripping a little from friday night.


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Offlineplate.82
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Re: I give up ;( :( :( ;( ;( [Re: Kinoko314]
    #28457785 - 09/03/23 10:04 PM (4 months, 22 days ago)

You seem so close! Based on your other results I wouldn't be too discouraged.

Spores on agar should only take about two weeks to get big enough for a transfer. If you're waiting several weeks that's a sign something isn't right. What does your SAB setup look like and how do you prep it for agar work?

What about your fruiting chamber?

Keep it up, shrooms are right around the corner.


Edited by plate.82 (09/03/23 10:05 PM)


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Invisiblestroopwafelman
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Re: I give up ;( :( :( ;( ;( [Re: wildlifesc]
    #28457931 - 09/04/23 05:19 AM (4 months, 21 days ago)

What kind of fruiting chamber did you use for the PF-Tek cakes? If the pic is taken in one I don't see the perlite (which is used in SGFC) or any water (used in a water tub) so perhaps they just dried out?


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Invisiblewildlifesc
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Re: I give up ;( :( :( ;( ;( [Re: stroopwafelman]
    #28458293 - 09/04/23 11:26 AM (4 months, 21 days ago)

Thank you for the encouragements.  I felt that I was close at some point, but then when I realized I could have wasted an entire year just growing mold and there is no way I can tell them apart, it seems that I am nowhere close at all..

This is what my passive tubs look like, I have different sizes.  I put water at the bottom and I mist it every 24~48 hours.  The one shown in the picture is dirty and dry because I gave up on the tub and didn't tend to it for a few weeks.  Usually I would mist it to keep condensation on the walls.



I also have an active tub where I have a circulating fan and a reptile fogger all controlled by an arduino to maintain humidity.

My SAB is just a regular upside down tote with two holes for hands like everyone else's.  Before I do SAB work I run a HEPA air filter for at least 12 hours to clean the air in the house, then I turn off central HVAC during the SAB work.  I wipe everything down with alcohol and wear a mask.


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Offlineplate.82
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Re: I give up ;( :( :( ;( ;( [Re: wildlifesc]
    #28458341 - 09/04/23 12:22 PM (4 months, 21 days ago)

The Arduino setup is a nice touch! I've wanted to do a similar project for a few years, and even bought most of the parts but never got around to mastering the coding.

Your SAB setup sounds OK. Make sure the filter isn't running while your working with the SAB as you want to eliminate any air currents. Do your work in a smaller enclosed space like a bathroom- I like to remove any towels or bath matts and allow the air to settle for at least 10 minutes after I turn the air filter off. I then begin wiping down any surfaces my SAB will come into contact with, sanitize my (gloved) hands with alcohol, followed my the SAB itself, and then all my materials. After I inoculate my petri dishes I store them individually in ziplock bags before removing them from the SAB.

I don't think your fruiting chamber is the main problem, and that can be adjusted as you go.

I say make some new plates and start a grow log. Post pictures once a week or so to get advice as you go. Even starting from spores you should have some shrooms by November.


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Invisiblestroopwafelman
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Re: I give up ;( :( :( ;( ;( [Re: wildlifesc]
    #28458362 - 09/04/23 12:41 PM (4 months, 21 days ago)

Just go wit the  Water Tub Tek, no holes, perhaps a slightly smaller tub since fruiting 2 cakes in what looks like 70? 80? quart tub is excessive, get a small grill rack like in the water tub tek, balance it on top of 4 1/2pint jars, pour a few inches of water on the bottom of the tub, a few days after placing the cakes in there flip the lid. Maintain surface conditions (tiny droplets on the surface) as per the tek after the mycelium colonizes the casing. No fanning. Done.


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Invisiblewildlifesc
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Re: I give up ;( :( :( ;( ;( [Re: stroopwafelman]
    #28458406 - 09/04/23 01:24 PM (4 months, 21 days ago)

I trashed the rest of my edible cultures.  Took a picture before they went bye-bye.  Do any of these look like mycelium?  Or are they all mold?



I'll start over with some spores shortly.  Hopefully they might fruit before it gets too cold (we keep the house around 66F in the winter).

I did have the GT cakes in a smaller tub than the one in the picture of my last post.  The GT tub is probably half the size of the one in the picture.


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Invisiblecovertjoy

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Re: I give up ;( :( :( ;( ;( [Re: wildlifesc]
    #28458415 - 09/04/23 01:30 PM (4 months, 21 days ago)

Quote:

wildlifesc said:
I trashed the rest of my edible cultures.  Took a picture before they went bye-bye.  Do any of these look like mycelium?  Or are they all mold?



I'll start over with some spores shortly.  Hopefully they might fruit before it gets too cold (we keep the house around 66F in the winter).

I did have the GT cakes in a smaller tub than the one in the picture of my last post.  The GT tub is probably half the size of the one in the picture.




They probably are mushroom mycelium since most molds will sporulate from the centre, changing color. Perhaps some molds are different, I am not sure. They don't look good though.


--------------------


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InvisiblePsil-y-nat-y
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Re: I give up ;( :( :( ;( ;( [Re: covertjoy]
    #28458491 - 09/04/23 02:56 PM (4 months, 21 days ago)

This gave me flashbacks to when I proudly showed my family what I thought was healthy oyster mushroom mycelium growing.


I only started questioning things after I saw green black and brown, also the weird vomit like thing. I cringe


1. What temperature do u store ur agar plates at?

2. When u ejaculated ur spores onto a fresh petri dish, are u able to only squirt one tiny drop out?

It's so much easier when u only have legit 1 tiny drop of spore water to watch grow.

When u spray ur syringe like u partaking in ur first interracial bukkake session, the growth that follows is too difficult to comprehend.


--------------------
Don't listen to any of my advice cuz I ended up with extreme HBP and nearly died. So I'm a literally an idiot. Don't let me guide u lol. I'm just trying to heal myself.. then I can be better for my family


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Invisiblewildlifesc
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Re: I give up ;( :( :( ;( ;( [Re: Psil-y-nat-y]
    #28458730 - 09/04/23 06:12 PM (4 months, 21 days ago)

1) I grow them in room temperature (~26C in the Summer) and then if I want to keep them for longer I throw them in the fridge in a ziplock bag (probably around ~7C)

2) I germinate from spore print.  For oyster and lions mane the spore print looks very thin and hard to see, so I scrape it with the scalpel tip and then just dip the tip into the agar to make sure some of it gets onto the agar.  For GT the spore chunks are large and visible so I just scrape some off the foil and see that they fall onto the agar.

Maybe I need to rub them across the plate after they drop to thin them out?  A lot of youtube instructions I see they just dump the spore onto the agar and call it a day.


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OfflineA.k.aM
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Re: I give up ;( :( :( ;( ;( [Re: wildlifesc]
    #28458749 - 09/04/23 06:29 PM (4 months, 21 days ago)

Why don’t you try knocking the cakes with spores instead of agar?


Your first plate picture of the mystery culture looks like it’s got knots. Maybe a rogue spore landed in there.


Anyway based on those plates I think you need to tighten up your SAB work. If your syringe germ plates have been looking clean using the shringe to knock the cakes up will eliminate the issue of getting contaminated while inoculating with agar in the SAB.


--------------------
LAGM2020


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OfflineKinoko314
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Re: I give up ;( :( :( ;( ;( [Re: wildlifesc] * 1
    #28458796 - 09/04/23 07:37 PM (4 months, 21 days ago)

Quote:

wildlifesc said:
1) I grow them in room temperature (~26C in the Summer) and then if I want to keep them for longer I throw them in the fridge in a ziplock bag (probably around ~7C)

2) I germinate from spore print.  For oyster and lions mane the spore print looks very thin and hard to see, so I scrape it with the scalpel tip and then just dip the tip into the agar to make sure some of it gets onto the agar.  For GT the spore chunks are large and visible so I just scrape some off the foil and see that they fall onto the agar.

Maybe I need to rub them across the plate after they drop to thin them out?  A lot of youtube instructions I see they just dump the spore onto the agar and call it a day.




I think the way you did it for GT could cause more contamination.  Maybe a lot more.

Whatever method you use, it's good to spread out the spores so it's not one big clump.  I think it struggles more when many different genetics from different germinations have to figure out how to work together. I'm not an expert though.

At the very least it will make it harder to isolate genetics.  It will be a jumble.  I made this mistake with some natalensis, and now I'm starting over.

YouTube is full of videos showing bad practices.  Don't trust everything you see over there.


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Invisiblewildlifesc
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Re: I give up ;( :( :( ;( ;( [Re: A.k.a]
    #28458823 - 09/04/23 08:07 PM (4 months, 21 days ago)

Quote:

A.k.a said:
Your first plate picture of the mystery culture looks like it’s got knots. Maybe a rogue spore landed in there.





Are you saying my contaminated plate that I didn't inoculate but started growing stuff might actually be somehow growing mushroom mycelium?  lol, I'll be damned.

I just inoculated a new plate with GT spore.  This time I only put a tiny spore chunk and then I tried to smear it out all over the plate.  We'll see how it goes...

BTW when I knocked the cakes with agar I used a syringe.  Injected some sterile water onto the agar, scraped the mycelium around, then sucked up the water to make an instant LC, then injected the LC into the cakes.  So I did not open up the cake jars to risk contamination.


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OfflineA.k.aM
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Re: I give up ;( :( :( ;( ;( [Re: wildlifesc]
    #28459112 - 09/05/23 03:46 AM (4 months, 20 days ago)

Ok I think that’s definitely the most likely vector then. Anything involving liquids has to be absolutely perfect, and since it seems like the SAB portion is where the problems are mixing up LI off a plate is pretty risky.


I mentioned trying the spore syringe straight to a cake because there’s no SAB work and a cake isn’t likely to contam during inoculation.  that’s why it was such a popular method, if the syringe is good it’s pretty foolproof.


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LAGM2020


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Offlinerocky_raccoon
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Re: I give up ;( :( :( ;( ;( [Re: A.k.a]
    #28459131 - 09/05/23 04:24 AM (4 months, 20 days ago)

Quote:

A.k.a said:
Why don’t you try knocking the cakes with spores instead of agar?





I would advise for the same. PF-Tek using spores seems to work sufficiently well, at least it did for me. You can later clone your best fruits to agar or grain. The goal is to get some clean culture in the first place.

Concerning pinning: My GTs seem to love pasteurized horse manure mixed with verm as a casing. I wash and drain the manure after pasteurizing to avoid excessive salts. But I would only use it after you established your clone, since manure contains (beneficial) bacteria.

Other cube varieties might work better in your setting. EQs for example were pinning willingly and repeatedly from a simple verm casing on a rye grain jar.

Don't give up yet, there are still options to try.


--------------------
Nothing in life is to be feared, it is only to be understood.
-Marie Curie



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Offlinephenyl
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Re: I give up ;( :( :( ;( ;( [Re: wildlifesc]
    #28459151 - 09/05/23 06:02 AM (4 months, 20 days ago)

Quote:

wildlifesc said:
Quote:

A.k.a said:
Your first plate picture of the mystery culture looks like it’s got knots. Maybe a rogue spore landed in there.





Are you saying my contaminated plate that I didn't inoculate but started growing stuff might actually be somehow growing mushroom mycelium?  lol, I'll be damned.






The more interesting question is how did it get in there to begin with and what else may have landed in other plates.


--------------------
The fool who persists in his folly will become wise.


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