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YoshiTrainer
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Re: Official Copelandia/Panaeolus Thread [Re: mikeybox] 3
#28191971 - 02/17/23 05:10 PM (11 months, 5 days ago) |
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Split the stipe, use sterile needle-nosed tweezers to snip out a section and transfer.
I think Adas has a proper write up in his signature too?
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mikeybox
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Re: Official Copelandia/Panaeolus Thread [Re: YoshiTrainer]
#28191977 - 02/17/23 05:14 PM (11 months, 5 days ago) |
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Well, I tried this with my pan cambo Goliath, except I used a sterilized scalpel to slice a bit of tissue from the center of a stem that I split open. I took 3 transfers to agar.
Unfortunately, all 3 are growing bacteria, and I'm not seeing mycelium. I think there was bacteria on the inside of the stem. Also the stem was hollow.
I wonder if there is still a chance mycelium might grow and outrun the bacteria? How long do pan clones usually take to see growth?
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ruawakeyet

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Re: Official Copelandia/Panaeolus Thread [Re: mikeybox] 1
#28191988 - 02/17/23 05:23 PM (11 months, 5 days ago) |
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They show pretty quick, usually within the first 24 hours. Next time, try it with water agar.
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YoshiTrainer
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Re: Official Copelandia/Panaeolus Thread [Re: ruawakeyet] 10
#28192024 - 02/17/23 05:57 PM (11 months, 5 days ago) |
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Needle-nose tweezers are a life saver! Here is an Alutacea clone that was started Feb 10. it is not Pan but had a stipe about the size of a toothpick. I like to make my clone and dirty agar transfers to the jar walls and have them grow to the agar, then transfer. It took days before my eyes could see growth on this one. I also made very small transfers to another jar from the top of the stipe where it connects to the cap. It took almost a week for me to see growth on that.
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Hindsight
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Re: Official Copelandia/Panaeolus Thread [Re: YoshiTrainer] 2
#28192038 - 02/17/23 06:11 PM (11 months, 5 days ago) |
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I do believe that bacteria grow inside the fruits. Quite often, even when I take a perfectly clean chunk out of a fat cube stipe - first splitting it in half with my hands, then using a sterile scalpel to cut a chunk out from the center, no part of which contacted the outside surface, I will get sneaky bacteria riding along. I don't always see it on the plate, but when I take a transfer from that plate and put it to LC the LC goes cloudy. Pans are harder because they are smaller but the trick is to use tweezers as others have mentioned, but also use them to pull out a long strand. Somewhere along that strand, the mycelium will almost always grow from.
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kirkeng
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Re: Official Copelandia/Panaeolus Thread [Re: Hindsight] 3
#28192057 - 02/17/23 06:24 PM (11 months, 5 days ago) |
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Damn Yoshi thatβs smart to use the side of the jar.. Iβm going to try that next time I get pan clones. My last bisporus clone grew a ton of bacteria but the mycelium eventually rose right over it and made it to the clean spot, tough little bugger. Hopefully my plates have enough glass on the side to try that..
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Baba Yaga
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Re: Official Copelandia/Panaeolus Thread [Re: mikeybox] 5
#28192062 - 02/17/23 06:30 PM (11 months, 5 days ago) |
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Quote:
mikeybox said: Has anybody successfully cloned pans? Seems a lot harder due to the stems being so much smaller vs. cubes...
If you have successfully cloned pans, can you give me advice how you did it?
I also recommend to take tissue to water agar. Bacteria gets suppressed and mycelium starts growing out almost instantly. You don't have to be very precise with your cuts either. Let the mycelium grow out at leas 30mm in diameter, take to a new regular plate and from there to LC. Worked very reliably for me.

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Scotay
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Re: Official Copelandia/Panaeolus Thread [Re: Baba Yaga]
#28192409 - 02/17/23 11:15 PM (11 months, 4 days ago) |
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MikeyB..... Hey man that looks great!!! Well done!! Nef.... Yours look great as well!!! Buncha panheads!!! I've got agar going right now with with the Bisbo Hopefully it'll pan out!!! Pun intended!! Happy Saturday friends and neighbors!! I hope everybodys tents tubs and trays are kickin' it!!! Peace and chicken grease!! πβοΈ
-------------------- Just chillin like a Villain and talking to like minded individuals!! Mush Love!! πβοΈ
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Adas
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Re: Official Copelandia/Panaeolus Thread [Re: Scotay] 1
#28192510 - 02/18/23 03:32 AM (11 months, 4 days ago) |
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Yoshi, amazing idea to stick it on the glass.
Hindsight, I haven't noticed bacteria growing on the inside of mushrooms. Many of my Pan clones were flawless from the get-go. Consider that your LCs may have gotten contaminated by other means than invisible bacteria riding along the mycelium. I've heard it can happen, at least with some species, but I haven't seen it with the species I've worked with.
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Hindsight
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Re: Official Copelandia/Panaeolus Thread [Re: Adas] 5
#28192617 - 02/18/23 07:20 AM (11 months, 4 days ago) |
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Yea Adas it was quite a surprise to me. I figured it was just with small fruits - like pans and semps and mexicana - I figured that they were small enough that I couldn't avoid getting at least some parts of the outer-exposed portions of the stipe in my tissue sample that then went to agar. But even with larger fruits, now that I am using LC for every single culture I work with, I am finding bacteria that previously went un-noticed even on agar. I have taken a meaty cube tissue sample, put it to plate, let it grow - looked clean, so put it to LC and it went slightly cloudy in a few days. Scoped it to find some bacteria. Put it to PF cake, let it grow out a bit, took a transfer from the cake to a plate, then transferred that to LC and it was flawless. Every time I do a cleanup on a PF cake and transfer it to LC, I get a clean LC 100% of the time (so far) which tells me my LC isn't getting contaminated by other means. But my sample size is not crazy high yet.... I've done around 8-12 PF -> LC cleanups so far. So yeah I do admit that there is a chance it is something I'm doing but I'm not sure what it could be.
But you know what, this gives me an idea for another experiment..... taking some clone tissues from pans and checking them under the scope for bacteria. Would have to do this on pans big enough to ensure I'm not touching the outside of the stipe, just to be certain. Shouldn't be too hard - the Bispos and Peace River I have grow pretty big. Could also try on cubes and semps.
On another note, the mystery of the Mystery South Africa pan cyans continues. As you may recall, my last set of trays just overlayed heavily and pushed out a few meager looking fruits around the very edges. I tried re-casing, and I tried scraping the casing off and running without casing - neither really worked. With this new round of trays, I added some leached HWFP to my normal pan sub of straw/verm/manure to see if perhaps they wanted wood. The results:

Even worse than the last trays. If you zoom way in you can see a few meager, unhappy fruits. I have one more tray of this that I just put in the tent, using a sub of hemp/verm/manure. My guess is it will probably do the same thing. I did clone a fruit from the last tray and had to clean it up on PF; it's now on LC and looking clean. I will use that clone culture for my future attempts, and I am going to abandon the tent for these and switch to the new bubbler/fogger tent I built (big tote with water and air stones in the bottom plus a fogger on a cycle timer to saturate every 12 hours). I'm also switching to 2 pint twistloc cakes for this species/variety until I figure out what it wants.
Edited by Hindsight (02/18/23 07:21 AM)
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Timestop413
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Re: Official Copelandia/Panaeolus Thread [Re: Baba Yaga]
#28192721 - 02/18/23 08:59 AM (11 months, 4 days ago) |
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Quote:
Baba Yaga said:
I also recommend to take tissue to water agar. Bacteria gets suppressed and mycelium starts growing out almost instantly. You don't have to be very precise with your cuts either. Let the mycelium grow out at leas 30mm in diameter, take to a new regular plate and from there to LC. Worked very reliably for me.


Are these clones including outer portions of the stem as well? How are people splitting open their stems since they are so small? With cubes..I can do by hand but should pans be done in a different way? I do have needle nose tweezers fwiw.
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Hindsight
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Re: Official Copelandia/Panaeolus Thread [Re: Timestop413] 1
#28192747 - 02/18/23 09:15 AM (11 months, 4 days ago) |
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Slice the very bottom end of the stem with a razor to create a starting point, then use fingers or tweezers to pull each half apart like string cheese. Re-sterilize tweezers and grab a fiber from the inside of the stem. Adas has a nice tek on it.
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Adas
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Re: Official Copelandia/Panaeolus Thread [Re: Hindsight] 1
#28192805 - 02/18/23 10:03 AM (11 months, 4 days ago) |
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Hindsight, that's really crazy. You might be on to something then. I just normally don't bother with LCs and my spawn is clean so it didn't occur to me this could be a possibility.
The SA Pan tubs look insane! Pretty nice to look at, despite the fact that it's not quite doing what you want it to
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Baba Yaga
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Re: Official Copelandia/Panaeolus Thread [Re: Timestop413] 1
#28192836 - 02/18/23 10:38 AM (11 months, 4 days ago) |
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Hindsight are you taking clones directly to BRF pucks? Just reminding me of bakedbeings 3 steps from spores to LC and thinking this could be used with clone tissue as well to reliably fast track the process.
Those SA are crazy.
Quote:
Timestop413 said:
Quote:
Baba Yaga said:
I also recommend to take tissue to water agar. Bacteria gets suppressed and mycelium starts growing out almost instantly. You don't have to be very precise with your cuts either. Let the mycelium grow out at leas 30mm in diameter, take to a new regular plate and from there to LC. Worked very reliably for me.


Are these clones including outer portions of the stem as well? How are people splitting open their stems since they are so small? With cubes..I can do by hand but should pans be done in a different way? I do have needle nose tweezers fwiw.
Yes, there is a pin pulled from a cake and the big chunky bits are stem segments including all the outer surface taken straight to WA.
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Hindsight
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Re: Official Copelandia/Panaeolus Thread [Re: Baba Yaga]
#28192850 - 02/18/23 10:51 AM (11 months, 4 days ago) |
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lol thanks for making me feel better Adas haha. I'll figure these mystery pans out eventually.
Baba, funny you should mention that because this week I've been thinking about starting to do that. It will skip the first step of agar. It adds some steps and time if the clone tissue sample was clean but as many as I've seen that are dirty, I feel like on average, it would save me time.
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mushroom_gnome
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Re: Official Copelandia/Panaeolus Thread [Re: Hindsight] 2
#28192880 - 02/18/23 11:16 AM (11 months, 4 days ago) |
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Contam or something different? These pancakes colonized fantastic with no seeming signs of contam. Fruits in multiple jars are expressing in this way.

-------------------- Mystery Mushroom
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old_fart_out_west
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Re: Official Copelandia/Panaeolus Thread [Re: mushroom_gnome]
#28193009 - 02/18/23 12:37 PM (11 months, 4 days ago) |
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Hey guys!
Didn't see anyone answer my question from a few days back, so was hoping someone could take a look and let me know their thoughts:
What causes pan cyan caps and stems to turn dark?

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Adas
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Gnome, I'm not sure but could be water issues?
Fart, stems can turn blue like that when misted. Sometimes Pans don't handle misting well.
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old_fart_out_west
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Re: Official Copelandia/Panaeolus Thread [Re: Adas]
#28193119 - 02/18/23 02:04 PM (11 months, 4 days ago) |
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Good to know, and makes sense considering I had a fairly heavy mist falling over them from my humidifier (1 min every 13 min; dial was turned up to a 5 out of 10 intensity).
How do you avoid the bluing of the stems and caps, while still retaining high humidity levels and ensuring the pans are properly watered?
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Adas
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I have personally noticed that sufficient FAE makes it possible to mist them without damage. At least most of the time. Just like how it works in nature. Your caps seem small so I think your FAE wasn't quite where it should be.
Hope this helps.
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